Compounds, Compositions and Methods for the Treatment of Beta-Amyloid Diseases and Synucleinopathies

ABSTRACT

Dihydroxyaryl compounds and pharmaceutically acceptable esters, their synthesis, pharmaceutical compositions containing them, and their use in the treatment of β-amyloid diseases, such as observed in Alzheimer&#39;s disease, and synucleinopathies, such as observed in Parkinson&#39;s disease, and the manufacture of medicaments for such treatment.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.provisional application Ser. Nos. 61/001,441, entitled “Compounds,Compositions and Methods for the Treatment of β-Amyloid Diseases andSynucleinopathies” to Esposito et al., filed Oct. 31, 2007.

TECHNICAL FIELD

This invention relates to bis-dihydroxyaryl compounds andpharmaceutically acceptable salts, their synthesis, pharmaceuticalcompositions containing them, and their use in the treatment of Aβamyloid disease, such as observed in Alzheimer's disease, andsynucleinopathies, such as observed in Parkinson's disease, and in themanufacture of medicaments for such treatment.

BACKGROUND OF THE INVENTION

Alzheimer's disease is characterized by the accumulation of a 39-43amino acid peptide termed the β-amyloid protein or Aβ, in a fibrillarform, existing as extracellular amyloid plaques and as amyloid withinthe walls of cerebral blood vessels. Fibrillar Aβ amyloid deposition inAlzheimer's disease is believed to be detrimental to the patient andeventually leads to toxicity and neuronal cell death, characteristichallmarks of Alzheimer's disease. Accumulating evidence implicatesamyloid, and more specifically, the formation, deposition, accumulationand/or persistence of Aβ fibrils, as a major causative factor ofAlzheimer's disease pathogenesis. In addition, besides Alzheimer'sdisease, a number of other amyloid diseases involve formation,deposition, accumulation and persistence of Aβ fibrils, including Down'ssyndrome, disorders involving congophilic angiopathy, such as but notlimited to, hereditary cerebral hemorrhage of the Dutch type, andcerebral β-amyloid angiopathy.

Parkinson's disease is another human disorder characterized by theformation, deposition, accumulation and/or persistence of abnormalfibrillar protein deposits that demonstrate many of the characteristicsof amyloid. In Parkinson's disease, an accumulation of cytoplasmic Lewybodies consisting of filaments of α-synuclein are believed important inthe pathogenesis and as therapeutic targets. New agents or compoundsable to inhibit α-synuclein formation, deposition, accumulation and/orpersistence, or disrupt pre-formed α-synuclein fibrils (or portionsthereof) are regarded as potential therapeutics for the treatment ofParkinson's and related synucleinopathies. A 35 amino acid fragment ofα-synuclein that has the ability to form amyloid-like fibrils either invitro or as observed in the brains of patients with Parkinson's disease.The fragment of α-synuclein is a relative important therapeutic targetas this portion of α-synuclein is believed crucial for formation of Lewybodies as observed in all patients with Parkinson's disease,synucleinopathies and related disorders. In addition, the α-synucleinprotein which forms fibrils, and is Congo red and Thioflavin S positive(specific stains used to detect amyloid fibrillar deposits), is found aspart of Lewy bodies in the brains of patients with Parkinson's disease,Lewy body disease (Lewy in Handbuch der Neurologie, M. Lewandowski, ed.,Springer, Berlin pp. 920-933, 1912; Pollanen et al, J. Neuropath. Exp.Neurol. 52:183-191, 1993; Spillantini et al, Proc. Natl. Acad. Sci. USA95:6469-6473, 1998; Arai et al, Neurosci. Lett. 259:83-86, 1999),multiple system atrophy (Wakabayashi et al, Acta Neuropath. 96:445-452,1998), dementia with Lewy bodies, and the Lewy body variant ofAlzheimer's disease. In Parkinson's disease, fibrils develop in thebrains of patients with this disease which are Congo red and ThioflavinS positive, and which contain predominant beta-pleated sheet secondarystructure.

Amyloid as a Therapeutic Target for Alzheimer's Disease

Alzheimer's disease also puts a heavy economic burden on society. Arecent study estimated that the cost of caring for one Alzheimer'sdisease patient with severe cognitive impairments at home or in anursing home, is more than $47,000 per year (A Guide to UnderstandingAlzheimer's Disease and Related Disorders). For a disease that can spanfrom 2 to 20 years, the overall cost of Alzheimer's disease to familiesand to society is staggering. The annual economic toll of Alzheimer'sdisease in the United States in terms of health care expenses and lostwages of both patients and their caregivers is estimated at $80 to $100billion (2003 Progress Report on Alzheimer's Disease).

Tacrine hydrochloride (“Cognex”), the first FDA approved drug forAlzheimer's disease, is a acetylcholinesterase inhibitor (Cutler andSramek, N. Engl. J. Med. 328:808 810, 1993). However, this drug hasshowed limited success in producing cognitive improvement in Alzheimer'sdisease patients and initially had major side effects such as livertoxicity. The second FDA approved drug, donepezil (“Aricept”), which isalso an acetylcholinesterase inhibitor, is more effective than tacrine,by demonstrating slight cognitive improvement in Alzheimer's diseasepatients (Barner and Gray, Ann. Pharmacotherapy 32:70-77, 1998; Rogersand Friedhoff, Eur. Neuropsych. 8:67-75, 1998), but is not believed tobe a cure. Therefore, it is clear that there is a need for moreeffective treatments for Alzheimer's disease patients.

Alzheimer's disease is characterized by the deposition and accumulationof a 39-43 amino acid peptide termed the beta-amyloid protein, Aβ orβ/A4 (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890, 1984;Masters et al., Proc. Natl. Acad. Sci. USA 82:4245-4249, 1985; Husby etal., Bull. WHO 71:105-108, 1993). Aβ is derived by protease cleavagefrom larger precursor proteins termed β-amyloid precursor proteins(APPs) of which there are several alternatively spliced variants. Themost abundant forms of the APPs include proteins consisting of 695, 751and 770 amino acids (Tanzi et al., Nature 31:528-530, 1988).

The small Aβ peptide is a major component that makes up the amyloiddeposits of “plaques” in the brains of patients with Alzheimer'sdisease. In addition, Alzheimer's disease is characterized by thepresence of numerous neurofibrillary “tangles”, consisting of pairedhelical filaments which abnormally accumulate in the neuronal cytoplasm(Grundke-Iqbal et al., Proc. Natl. Acad. Sci. USA 83:4913-4917, 1986;Kosik et al., Proc. Natl. Acad. Sci. USA 83:4044-4048, 1986; Lee et al.,Science 251:675-678, 1991). The pathological hallmark of Alzheimer'sdisease is therefore the presence of “plaques” and “tangles”, withβ-amyloid being deposited in the central core of the plaques. The othermajor type of lesion found in the Alzheimer's disease brain is theaccumulation of β-amyloid in the walls of blood vessels, both within thebrain parenchyma and in the walls of meningeal vessels that lie outsidethe brain. The β-amyloid deposits localized to the walls of bloodvessels are referred to as cerebrovascular amyloid or congophilicangiopathy (Mandybur, J. Neuropath. Exp. Neurol. 45:79-90, 1986;Pardridge et al., J. Neurochem. 49:1394-1401, 1987)

For many years there has been an ongoing scientific debate as to theimportance of “β-amyloid” in Alzheimer's disease, and whether the“plaques” and “tangles” characteristic of this disease were a cause ormerely a consequence of the disease. Within the last few years, studiesnow indicate that β-amyloid is indeed a causative factor for Alzheimer'sdisease and should not be regarded as merely an innocent bystander. TheAlzheimer's Aβ protein in cell culture has been shown to causedegeneration of nerve cells within short periods of time (Pike et al.,Br. Res. 563:311-314, 1991; J. Neurochem. 64:253-265, 1995). Studiessuggest that it is the fibrillar structure (consisting of a predominantβ-pleated sheet secondary structure), which is responsible for theneurotoxic effects. Aβ has also been found to be neurotoxic in slicecultures of hippocampus (Harrigan et al., Neurobiol. Aging 16:779-789,1995) and induces nerve cell death in transgenic mice (Games et al.,Nature 373:523-527, 1995; Hsiao et al., Science 274:99-102, 1996).Injection of the Alzheimer's Aβ into rat brain also causes memoryimpairment and neuronal dysfunction (Flood et al., Proc. Natl. Acad.Sci. USA 88:3363-3366, 1991; Br. Res. 663:271-276, 1994).

Probably, the most convincing evidence that Aβ amyloid is directlyinvolved in the pathogenesis of Alzheimer's disease comes from geneticstudies. It was discovered that the production of Aβ can result frommutations in the gene encoding, its precursor, β-amyloid precursorprotein (Van Broeckhoven et al., Science 248:1120-1122, 1990; Murrell etal., Science 254:97-99, 1991; Haass et al., Nature Med. 1:1291-1296,1995). The identification of mutations in the beta-amyloid precursorprotein gene that cause early onset familial Alzheimer's disease is thestrongest argument that amyloid is central to the pathogenetic processunderlying this disease. Four reported disease-causing mutations havebeen discovered which demonstrate the importance of Aβ in causingfamilial Alzheimer's disease (reviewed in Hardy, Nature Genet.1:233-234, 1992). All of these studies suggest that providing a drug toreduce, eliminate or prevent fibrillar Aβ formation, deposition,accumulation and/or persistence in the brains of human patients willserve as an effective therapeutic.

Parkinson's Disease and Synucleinopathies

Parkinson's disease is a neurodegenerative disorder that ispathologically characterized by the presence of intracytoplasmic Lewybodies (Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer,Berlin, pp. 920-933, 1912; Pollanen et al., J. Neuropath. Exp. Neurol.52:183-191, 1993), the major components of which are filamentsconsisting of α-synuclein (Spillantini et al., Proc. Natl. Acad. Sci.USA 95:6469-6473, 1998; Arai et al., Neurosci. Lett. 259:83-86, 1999),an 140-amino acid protein (Ueda et al., Proc. Natl. Acad. Sci. USA90:11282-11286, 1993). Two dominant mutations in α-synuclein causingfamilial early onset Parkinson's disease have been described suggestingthat Lewy bodies contribute mechanistically to the degeneration ofneurons in Parkinson's disease and related disorders (Polymeropoulos etal., Science 276:2045-2047, 1997; Kruger et al., Nature Genet.18:106-108, 1998). Recently, in vitro studies have demonstrated thatrecombinant α-synuclein can indeed form Lewy body-like fibrils (Conwayet al., Nature Med. 4:1318-1320, 1998; Hashimoto et al., Brain Res.799:301-306, 1998; Nahri et al., J. Biol. Chem. 274:9843-9846, 1999).Most importantly, both Parkinson's disease-linked α-synuclein mutationsaccelerate this aggregation process, demonstrating that such in vitrostudies may have relevance for Parkinson's disease pathogenesis.Alpha-synuclein aggregation and fibril formation fulfills the criteriaof a nucleation-dependent polymerization process (Wood et al., J. Biol.Chem. 274:19509-19512, 1999). In this regard α-synuclein fibrilformation resembles that of Alzheimer's β-amyloid protein (AβP) fibrils.Alpha-synuclein recombinant protein, and non-Aβ component (known asNAC), which is a 35-amino acid peptide fragment of α-synuclein, bothhave the ability to form fibrils when incubated at 37° C., and arepositive with amyloid stains such as Congo red (demonstrating ared/green birefringence when viewed under polarized light) andThioflavin S (demonstrating positive fluorescence) (Hashimoto et al.,Brain Res. 799:301-306, 1998; Ueda et al., Proc. Natl. Acad. Sci. USA90:11282-11286, 1993).

Synucleins are a family of small, presynaptic neuronal proteins composedof α-, β-, and γ-synucleins, of which only α-synuclein aggregates havebeen associated with several neurological diseases (Ian et al., ClinicalNeurosc. Res. 1:445-455, 2001; Trojanowski and Lee, Neurotoxicology23:457-460, 2002). The role of synucleins (and in particular,alpha-synuclein) in the etiology of a number of neurodegenerativediseases has developed from several observations. Pathologically,synuclein was identified as a major component of Lewy bodies, thehallmark inclusions of Parkinson's disease, and a fragment thereof wasisolated from amyloid plaques of a different neurological disease,Alzheimer's disease. Biochemically, recombinant α-synuclein was shown toform fibrils that recapitulated the ultrastructural features ofalpha-synuclein isolated from patients with dementia with Lewy bodies,Parkinson's disease and multiple system atrophy. Additionally, theidentification of mutations within the synuclein gene, albeit in rarecases of familial Parkinson's disease, demonstrated an unequivocal linkbetween synuclein pathology and neurodegenerative diseases. The commoninvolvement of α-synuclein in a spectrum of diseases such as Parkinson'sdisease, dementia with Lewy bodies, multiple system atrophy and the Lewybody variant of Alzheimer's disease has led to the classification ofthese diseases under the umbrella term of “synucleinopathies”.

Parkinson's disease α-synuclein fibrils, and the Aβ fibrils ofAlzheimer's disease, both consist of a predominantly β-pleated sheetstructure. Compounds found to inhibit Alzheimer's disease Aβ amyloidfibril formation have also been shown to be effective in the inhibitionof α-synuclein fibril formation, as illustrated in the Examples of thepresent invention. These compounds would therefore also serve astherapeutics for Parkinson's disease and other synucleinopathies, inaddition to having efficacy as a therapeutic for Alzheimer's disease.

Parkinson's disease and Alzheimer's disease are characterized by theinappropriate accumulation of insoluble aggregates comprised primarilyof misfolded proteins that are enriched in β-pleated sheet secondarystructure (reviewed in Cohen et al., Nature 426:905-909, 2003; Chiti etal., Annu. Rev. Biochem., 75:333-366, 2006). In Parkinson's disease,α-synuclein is the major constituent of these aggregates, as part ofLewy Bodies, and mutations in α-synuclein that increase its propensityto misfold and aggregate are observed in familial Parkinson's disease(Polymeropoulos et al., Science 276:1197-1199, 1997; Papadimitriou etal., Neurology 52:651-654, 1999).

Mitochondrial dysfunction, specifically as a result of impairment atcomplex I of the electron transport chain, is also a common feature ofParkinson's disease (Schapira et al., J. Neurochem., 54:823-827, 1990;reviewed in Greenamyre et al., IUBMB Life, 52:135-141, 2001). Directevidence for mitochondrial deficits in the etiology of Parkinson'sdisease came first from the observation that MPP+(1-methyl-4-phenyl-2,3-dihydropyridinium), the active metabolite of theparkinsonism toxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),inhibits complex I (Nicklas et al., Life Sci., 36:2503-2508, 1985).Subsequently, rotenone, another complex I inhibitor, was shown to be animproved model for α-synuclein aggregation because it reproduces theabove-mentioned α-synuclein-positive intracytoplasmic aggregates, inaddition to the behavioral changes and loss of dopaminergic neurons seenin the MPTP model. Rotenone toxicity of this type is seen in multiplemodel systems including rats (Betarbet et al., Nat. Neurosci.,3:1301-1306, 2000; Panov et al., J. Biol. Chem., 280:42026-42035, 2005),rat brain slices (Sherer et al., J. Neurosci., 23:10756-10764, 2003;Testa et al., Mol. Brain. Res., 134:109-118, 2005), C. elegans (Ved etal., J. Biol. Chem., 280:42655-42668, 2005) and cultured cells (Shereret al., J. Neurosci., 22:7006-7015, 2002) and has been shown to be aconsequence of increased oxidative damage resulting from complex Iinhibition.

To better understand the relationship of oxidative damage to mutantα-synuclein pathogenesis, a neuroblastoma cell line (using BE-M17 cells)has been established in the art that overexpresses A53T α-synuclein. Inthese cells, A53T α-synuclein aggregates in response to a variety ofoxidative stress-inducing agents and potentiates mitochondrialdysfunction and cell death (Ostrerova-Golts et al., J. Neurosci.,20:6048-6054, 2000). These cells are amenable to rotenone treatment asan oxidative stress inducer and hence, are particularly useful fortesting agents that might inhibit α-synucleinaggregation/fibrillogenesis.

Discovery and identification of new compounds or agents as potentialtherapeutics to arrest amyloid formation, deposition, accumulationand/or persistence that occurs in Alzheimer's disease, and Parkinson'sdisease, are desperately sought.

SUMMARY OF THE INVENTION

This invention relates to bis-dihydroxyaryl compounds andpharmaceutically acceptable salts thereof. The compounds are useful inthe treatment of β-amyloid diseases and synucleinopathies.

The compounds are:

compounds of the formula:

where:where R₁ and R₂, and R₃ and R₄ are hydroxyl groups independentlypositioned at one of the positions selected from the group consisting of2,3; 2,4; 2,5; 2,6; 3,5; 3,6; 4,5; 4,6 and 5,6, and R is selected from asulfonamide, heteroaryl, tricycloalkyl and —C(O)NR′ where R′ is selectedfrom H or CH₃ or pharmaceutically acceptable esters or salts thereof.

Also provided are any pharmaceutically-acceptable derivatives, includingsalts, esters, enol ethers or esters, acetals, ketals, orthoesters,hemiacetals, hemiketals, solvates, hydrates or prodrugs of thecompounds. Pharmaceutically-acceptable salts, include, but are notlimited to, amine salts, such as but not limited toN,N′-dibenzylethylenediamine, chloroprocaine, choline, ammonia,diethanolamine and other hydroxyalkylamines, ethylenediamine,N-methylglucamine, procaine, N-benzylphenethylamine,1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethylbenzimidazole, diethylamineand other alkylamines, piperazine, tris(hydroxymethyl)aminomethane,alkali metal salts, such as but not limited to lithium, potassium andsodium, alkali earth metal salts, such as but not limited to barium,calcium and magnesium, transition metal salts, such as but not limitedto zinc and other metal salts, such as but not limited to sodiumhydrogen phosphate and disodium phosphate, and also including, but notlimited to, salts of mineral acids, such as but not limited tohydrochlorides and sulfates, salts of organic acids, such as but notlimited to acetates, lactates, malates, tartrates, citrates, ascorbates,succinates, butyrates, valerates and fumarates.

Pharmaceutical formulations for administration by an appropriate routeand means containing effective concentrations of one or more of thecompounds provided herein or pharmaceutically acceptable derivatives,such as salts, esters, enol ethers or esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, solvates, hydrates or prodrugs, ofthe compounds that deliver amounts effective for the treatment ofamyloid diseases, are also provided.

The formulations are compositions suitable for administration by anydesired route and include solutions, suspensions, emulsions, tablets,dispersible tablets, pills, capsules, powders, dry powders forinhalation, sustained release formulations, aerosols for nasal andrespiratory delivery, patches for transdermal delivery and any othersuitable route. The compositions should be suitable for oraladministration, parenteral administration by injection, includingsubcutaneously, intramuscularly or intravenously as an injectableaqueous or oily solution or emulsion, transdermal administration andother selected routes.

Methods using such compounds and compositions for disrupting,disaggregating and causing removal, reduction or clearance of β-amyloidor α-synuclein fibrils are provided thereby providing new treatments forβ-amyloid diseases and synucleinopathies.

Also provided are methods for treatment, prevention or amelioration ofone or more symptoms of amyloid diseases or amyloidoses, including butnot limited to diseases associated with the formation, deposition,accumulation, or persistence of β-amyloid fibrils.

Methods for treatment of amyloid diseases, include, but are not limitedto Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhagewith amyloidosis of the Dutch type, and cerebral β-amyloid angiopathy.

Also provided are methods for treatment, prevention or amelioration ofone or more symptoms of synuclein diseases or synucleinopathies. In oneembodiment, the methods inhibit or prevent α-synuclein fibril formation,inhibit or prevent α-synuclein fibril growth, and/or cause disassembly,disruption, and/or disaggregation of preformed α-synuclein fibrils andα-synuclein-associated protein deposits. Synuclein diseases include, butare not limited to Parkinson's disease, familial Parkinson's disease,Lewy body disease, the Lewy body variant of Alzheimer's disease,dementia with Lewy bodies, multiple system atrophy, and theParkinsonism-dementia complex of Guam.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A shows several circular dichroism spectra illustrating thatAlzheimer's disease Aβ fibrils are disrupted by the compounds tested at1:1 wt/wt. FIG. 1B shows graphically the % inhibition.

FIG. 2 shows comparative circular dichroism spectra illustratingα-synuclein forms β-sheet rich structure after 4 days of agitation at37° C.

FIG. 3 shows several circular dichroism spectra illustrating that testedcompounds inhibit α-synuclein aggregation at 1:1 wt/wt. FIG. 3B showsgraphically the % inhibition.

FIG. 4 shows several circular dichroism spectra illustrating thatcompounds inhibit α-synuclein aggregation at 1:0.1 wt/wt. FIG. 4B showsgraphically the % inhibition.

FIG. 5 graphically summarizes the results, as measured by Thio T, of thetested compounds to inhibit Aβ fibril formation.

FIG. 6 graphically summarizes the results, as measured by Congo Red, ofthe tested compounds to inhibit Aβ fibril formation.

FIG. 7 graphically summarizes the results, as measured by Thio T, of thetested compounds to inhibit α-synuclein fibril formation.

FIG. 8 graphically summarizes the results, as measured by Congo Red, ofthe tested compounds to inhibit α-synuclein fibril formation.

FIGS. 9 A-D are examples of fluorescent photomicrographs demonstratingeffects of rotenone on number of thioflavin S-positive aggregates. FIG.9A is vehicle alone, FIG. 9B is 1 μM rotenone, FIG. 9C is 5 μM at lowmagnification, and FIG. 9D is 5 μM at high magnification. FIG. 9Esummarizes the quantitative analysis of the Thioflavin S in response torotenone treatment.

FIGS. 10 A-C are examples of fluorescent photomicrographs demonstratinga reduction in thioflavin S-positive aggregates (green fluorescence)upon application of a positive control compound. FIG. 10 A is untreated,FIG. 10 B shows 500 ng/mL of positive control compound and FIG. 10 Cshows 1 μg/mL positive control compound. FIG. 10D summarizes thequantitative analysis of dose dependent reduction in aggregation.

FIGS. 11 A-D are examples of fluorescent photomicrographs demonstratingthe effects of compound 1 on the presence of rotenone-induced thioflavinS-positive aggregates (green) in cells in a dose dependent manner. FIG.11 A is untreated (rotenone only), and FIGS. 11 B-D, respectively, show500 ng/mL, 1 μg/mL and 2 μg/mL of compound 1. FIG. 11D summarizes thequantitative analysis of the effects of the compound 1.

FIGS. 12 A-D are examples of fluorescent photomicrographs demonstratingthat compound 2 strongly reduces the presence of rotenone-inducedthioflavin S-positive aggregates (green) in cells. FIG. 12 A isuntreated (rotenone only), and FIGS. 12 B-D, respectively, show 500ng/mL, 1 μg/mL and 2 μg/mL of compound 2. FIG. 12E summarizes thequantitative analysis of the anti-aggregation effects of compound 2.

FIGS. 13 A-D are examples of fluorescent photomicrographs demonstratingthat compound 3 reduces the presence of rotenone-induced thioflavinS-positive aggregates (green) in cells in a dose dependent manner. FIG.13 A is untreated (rotenone only), and FIGS. 13 B-D, respectively, show500 ng/mL, 1 μg/mL and 2 μg/mL of compound 3. FIG. 13 E summarizes thequantitative analysis of the anti-aggregation effects of compound 3.

FIGS. 14 A-D are examples of fluorescent photomicrographs demonstratingthat compound 4 minimally reduces the presence of rotenone-inducedthioflavin S-positive aggregates (green) in cells in a dose dependentmanner. FIG. 14 A is untreated (rotenone only), and FIGS. 14 B-D,respectively, show 500 ng/mL, 1 μg/mL and 2 μg/mL of compound 4. FIG. 14E summarizes the quantitative analysis of the effects of compound 4.

FIGS. 15 A-D are examples of fluorescent photomicrographs demonstratingthat compound 5 mildly reduces the presence of rotenone-inducedthioflavin S-positive aggregates (green) in cells in a dose dependentmanner. FIG. 15 A is untreated (rotenone only), and FIGS. 15 B-D,respectively, show 500 ng/mL, 1 μg/mL and 2 μg/mL of compound 5. FIG. 15E summarizes the quantitative analysis of the anti-aggregation effectsof compound 5.

FIGS. 16 A-D are examples of fluorescent photomicrographs demonstratingthat compound 6 minimally affects the presence of rotenone-inducedthioflavin S-positive aggregates (green) in cells in a dose dependentmanner. FIG. 16 A is untreated (rotenone only), and FIGS. 16 B-D,respectively, show 500 ng/mL, 1 μg/mL and 2 μg/mL of compound 6. FIG. 16E summarizes the quantitative analysis of the effects of compound 6.

FIGS. 17 A-C are examples of fluorescent photomicrographs demonstratingthat compound 7 moderately reduces the presence of rotenone-inducedthioflavin S-positive aggregates (green) in cells in a dose dependentmanner. FIG. 17 A is untreated (rotenone only), and FIGS. 17 B-Crespectively show 500 ng/mL, and 2 μg/mL of compound 7. FIG. 17 Dsummarizes the quantitative analysis of the anti-aggregation effects ofcompound 7.

FIGS. 18 A-D are examples of fluorescent photomicrographs demonstratingthat compound 8 moderately reduces the presence of rotenone-inducedthioflavin S-positive aggregates (green) in cells in a dose dependentmanner. FIG. 18 A is untreated (rotenone only), and FIGS. 18 B-D,respectively, show 500 ng/mL, 1 μg/mL and 2 μg/mL of compound 8. FIG. 18E summarizes the quantitative analysis of the anti-aggregation effectsof compound 8.

FIGS. 19 A-D are examples of fluorescent photomicrographs demonstratingthat compound 9 reduces the presence of rotenone-induced thioflavinS-positive aggregates (green) in cells in a dose dependent manner. FIG.19 A is untreated (rotenone only), and FIGS. 19 B-D, respectively, show500 ng/mL, 1 μg/mL and 2 μg/mL of compound 9. FIG. 19 E summarizes thequantitative analysis of the anti-aggregation effects of compound 9where * p<0.05 relative to 1 μM rotenone only.

FIG. 20 is a graph showing 35-45% reduction in cell viability after 2days of treatment with rotenone as measured by the XTT Cytotoxicityassay.

FIG. 21 is a graph showing the ability of the positive control compoundto inhibit rotenone-induced toxicity as measured by the XTT Cytotoxicityassay.

FIG. 22 A is a graph showing that compound 1 is non-toxic up to 10μg/ml. FIG. 22 B is a graph showing the ability of compound 1 to protectagainst rotenone-induced toxicity as measured by the XTT Cytotoxicityassay.

FIG. 23 A is a graph showing that compound 2 is non-toxic up to 25μg/ml. FIG. 23 B is a graph showing the ability of compound 2 to protectagainst rotenone-induced toxicity as measured by the XTT Cytotoxicityassay.

FIG. 24 A is a graph showing that compound 3 is non-toxic up to 50μg/ml. FIG. 24 B is a graph showing the ability of compound 3 to protectagainst rotenone-induced toxicity as measured by the XTT Cytotoxicityassay.

FIG. 25 A is a graph showing that compound 4 is non-toxic up to 25μg/ml. FIG. 25 B is a graph showing the ability of compound 4 to protectagainst rotenone-induced toxicity as measured by the XTT Cytotoxicityassay.

FIG. 26 A is a graph showing that compound 5 is non-toxic up to 25μg/ml. FIG. 26 B is a graph showing the inability of compound 5 toprotect against rotenone-induced toxicity as measured by the XTTCytotoxicity assay.

FIG. 27 A is a graph showing that compound 6 is non-toxic up to 50μg/ml. FIG. 27 B is a graph showing the ability of compound 6 to protectagainst rotenone-induced toxicity as measured by the XTT Cytotoxicityassay.

FIG. 28 A is a graph showing that compound 7 is non-toxic up to 50μg/ml. FIG. 28 B is a graph showing the ability of compound 7 to protectagainst rotenone-induced toxicity as measured by the XTT Cytotoxicityassay.

FIG. 29 A is a graph showing that compound 8 is non-toxic up to 25μg/ml. FIG. 29 B is a graph showing the ability of compound 8 to protectagainst rotenone-induced toxicity as measured by the XTT Cytotoxicityassay.

FIG. 30 A is a graph showing that compound 9 is non-toxic up to 25μg/ml. FIG. 30 B is a graph showing the inability of compound 9 toprotect against rotenone-induced toxicity as measured by the XTTCytotoxicity assay.

FIG. 31 is a graph showing beam traversal times and the effects ofcompound treatment. Treatment with compounds 2 and 7 improve the motorperformance in the beam traversal test. At three months of treatment,compound 2 improves the motor performance (measured by a reduction intime to cross) in the beam traversal test significantly (p<0.05) by 49%,relative to vehicle-treated mice at the same age. At six months oftreatment, compound 7 improves the motor performance in the beamtraversal test significantly (p<0.05) by 35%, relative tovehicle-treated mice at the same age. In addition, compound 7 shows ageneral trend in improving motor performance by 39% at three months oftreatment, relative to vehicle-treated mice at the same age.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In this application, the following terms shall have the followingmeanings, without regard to whether the terms are used variantlyelsewhere in the literature or otherwise in the known art.

As used herein “Amyloid diseases” or “amyloidoses” are diseasesassociated with the formation, deposition, accumulation, or persistenceof Aβ amyloid fibrils. Such diseases include, but are not limited toAlzheimer's disease, Down's syndrome, hereditary cerebral hemorrhagewith amyloidosis of the Dutch type, and cerebral β-amyloid angiopathy.

As used herein, “Synuclein diseases” or “synucleinopathies” are diseasesassociated with the formation, deposition, accumulation, or persistenceof α-synuclein fibrils. Such diseases include, but are not limited toParkinson's disease, familial Parkinson's disease, Lewy body disease,the Lewy body variant of Alzheimer's disease, dementia with Lewy bodies,multiple system atrophy, and the Parkinsonism-dementia complex of Guam.

“Fibrillogenesis” refers to the formation, deposition, accumulationand/or persistence of β-amyloid fibrils, filaments, inclusions,deposits, as well as α-synuclein fibrils, filaments, inclusions,deposits or the like.

“Inhibition of fibrillogenesis” refers to the inhibition of formation,deposition, accumulation and/or persistence of such a β-amyloid fibrilsor α-synuclein fibril-like deposits.

“Disruption of fibrils or fibrillogenesis” refers to the disruption ofpre-formed β-amyloid or α-synuclein fibrils, that usually exist in apre-dominant β-pleated sheet secondary structure. Such disruption bycompounds provided herein may involve marked reduction or disassembly ofamyloid or synuclein fibrils as assessed by various methods such asThioflavin T fluorometry, Congo red binding, circular dichroism spectra,thioflavin S and cell based assays such as α-synuclein aggregation andXTT cytotoxicity assays and as demonstrated by the Examples presented inthis application.

“Neuroprotection” or “neuroprotective” refers to the ability of acompound to protect, reduce, alleviate, ameliorate, and/or attenuatedamage to nerve cells (neurodegeneration).

“Mammal” includes both humans and non-human mammals, such as companionanimals (cats, dogs, and the like), laboratory animals (such as mice,rats, guinea pigs, and the like) and farm animals (cattle, horses,sheep, goats, swine, and the like).

“Pharmaceutically acceptable excipient” means an excipient that isconventionally useful in preparing a pharmaceutical composition that isgenerally safe, non-toxic, and desirable, and includes excipients thatare acceptable for veterinary use or for human pharmaceutical use. Suchexcipients may be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

A “therapeutically effective amount” means the amount that, whenadministered to a subject or animal for treating a disease, issufficient to affect the desired degree of treatment, prevention orsymptom amelioration for the disease. A “therapeutically effectiveamount” or a “therapeutically effective dosage” in certain embodimentsinhibits, reduces, disrupts, disassembles β-amyloid or α-synucleinfibril formation, deposition, accumulation and/or persistence, ortreats, prevents, or ameliorates one or more symptoms of a diseaseassociated with these conditions, such as an amyloid disease or asynucleinopathy, in a measurable amount in one embodiment, by at least20%, in other embodiment, by at least 40%, in other embodiment by atleast 60%, and in still other embodiment by at least 80%, relative to anuntreated subject. Effective amounts of a compound provided herein orcomposition thereof for treatment of a mammalian subject are about 0.1to about 1000 mg/Kg of body weight of the subject/day, such as fromabout 1 to about 100 mg/Kg/day, in other embodiment, from about 10 toabout 500 mg/Kg/day. A broad range of disclosed composition dosages arebelieved to be both safe and effective.

The term “sustained release component” is defined herein as a compoundor compounds, including, but not limited to, polymers, polymer matrices,gels, permeable membranes, liposomes, microspheres, or the like, or acombination thereof, that facilitates the sustained release of theactive ingredient.

If the complex is water-soluble, it may be formulated in an appropriatebuffer, for example, phosphate buffered saline, or other physiologicallycompatible solutions. Alternatively, if the resulting complex has poorsolubility in aqueous solvents, then it may be formulated with anon-ionic surfactant such as Tween, or polyethylene glycol. Thus, thecompounds and their physiological solvents may be formulated foradministration by inhalation or insufflation (either through the mouthor the nose) or oral, buccal, parenteral, or rectal administration, asexamples.

As used herein, pharmaceutically acceptable derivatives of a compoundinclude salts, esters, enol ethers, enol esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, solvates, hydrates or prodrugsthereof. Such derivatives may be readily prepared by those of skill inthis art using known methods for such derivatization. The compoundsproduced may be administered to animals or humans without substantialtoxic effects and either are pharmaceutically active or are prodrugs.Pharmaceutically acceptable salts include, but are not limited to, aminesalts, such as but not limited to N,N′-dibenzylethylenediamine,chloroprocaine, choline, ammonia, diethanolamine and otherhydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine,N-benzylphenethylamine,1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethyl-benzimidazole, diethylamineand other alkylamines, piperazine and tris(hydroxymethyl)aminomethane;alkali metal salts, such as but not limited to lithium, potassium andsodium; alkali earth metal salts, such as but not limited to barium,calcium and magnesium; transition metal salts, such as but not limitedto zinc; and other metal salts, such as but not limited to sodiumhydrogen phosphate and disodium phosphate; and also including, but notlimited to, salts of mineral acids, such as but not limited tohydrochlorides and sulfates; and salts of organic acids, such as but notlimited to acetates, lactates, malates, tartrates, citrates, ascorbates,succinates, butyrates, valerates and fumarates. Pharmaceuticallyacceptable esters include, but are not limited to, alkyl, alkenyl,alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl andheterocyclyl esters of acidic groups, including, but not limited to,carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids,sulfinic acids and boronic acids. Pharmaceutically acceptable enolethers include, but are not limited to, derivatives of formula C═C(OR)where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,heteroaralkyl, cycloalkyl or heterocyclyl. Pharmaceutically acceptableenol esters include, but are not limited to, derivatives of formulaC═C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl,heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl.Pharmaceutically acceptable solvates and hydrates are complexes of acompound with one or more solvent or water molecules, or 1 to about 100,or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.

As used herein, treatment means any manner in which one or more of thesymptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. Treatment of a disease also includes preventingthe disease from occurring in a subject that may be predisposed to thedisease but does not yet experience or exhibit symptoms of the disease(prophylactic treatment), inhibiting the disease (slowing or arrestingits development), providing relief from the symptoms or side-effects ofthe disease (including palliative treatment), and relieving the disease(causing regression of the disease), such as by disruption of pre-formedβ-amyloid or α-synuclein fibrils.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular compound or pharmaceutical compositionrefers to any lessening, whether permanent or temporary, lasting ortransient that can be attributed to or associated with administration ofthe composition.

As used herein, inhibition of α-synuclein fibril formation, deposition,accumulation, aggregation, and/or persistence is believed to beeffective treatment for a number of diseases involving α-synuclein, suchas Parkinson's disease, Lewy body disease and multiple system atrophy.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized by one or more steps or processes orotherwise converted to the biologically, pharmaceutically ortherapeutically active form of the compound. To produce a prodrug, thepharmaceutically active compound is modified such that the activecompound will be regenerated by metabolic processes. The prodrug may bedesigned to alter the metabolic stability or the transportcharacteristics of a drug, to mask side effects or toxicity, to improvethe flavor of a drug or to alter other characteristics or properties ofa drug. By virtue of knowledge of pharmacodynamic processes and drugmetabolism in vivo, those of skill in this art, once a pharmaceuticallyactive compound is known, can design prodrugs of the compound (see,e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, OxfordUniversity Press, New York, pages 388-392).

Chemical structures for some of the compounds of this invention areshown. The names of the compounds are variously IUPAC names [namesderived according to the accepted IUPAC (International Union of Pure andApplied Chemistry) system established by the coalition of the Commissionon Nomenclature of Organic Chemistry and the Commission on PhysicalOrganic Chemistry, as can be found at http://www.chem.qmul.ac.uk/iupac],names derived from IUPAC names by addition or substitution (for example,by the use of “3,4-methylenedioxyphenyl” derived from “phenyl” insteadof “benzo[1,3]dioxol-5-yl”), and names derived from the names ofreactants (for example, by the use of “3,4-dihydroxybenzoic acid3,4-dihydroxyanilide” instead of“N-(3,4-dihydroxyphenyl)-3,4-dihydroxybenzamide”). However, the namesused are explicitly equated to chemical structures, and are believed tobe readily understood by a person of ordinary skill in the art.

“A pharmaceutical agent” or “pharmacological agent” or “pharmaceuticalcomposition” refers to a compound or combination of compounds used fortreatment, preferably in a pure or near pure form. In the specification,pharmaceutical or pharmacological agents include the compounds of thisinvention. The compounds are desirably purified to 80% homogeneity, andpreferably to 90% homogeneity. Compounds and compositions purified to99.9% homogeneity are believed to be advantageous. As a test orconfirmation, a suitable homogeneous compound on HPLC would yield, whatthose skilled in the art would identify as a single sharp-peak band.

It is to be understood that the compounds provided herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or be stereoisomeric ordiastereomeric mixtures. In the case of amino acid residues, suchresidues may be of either the L- or D-form. The configuration fornaturally occurring amino acid residues is generally L. When notspecified the residue is the L form. As used herein, the term “aminoacid” refers to α-amino acids which are racemic, or of either the D- orL-configuration. The designation “d” preceding an amino acid designation(e.g., dAla, dSer, dVal, etc.) refers to the D-isomer of the amino acid.The designation “dl” preceding an amino acid designation (e.g., dlPip)refers to a mixture of the L- and D-isomers of the amino acid. It is tobe understood that the chiral centers of the compounds provided hereinmay undergo epimerization in vivo. As such, one of skill in the art willrecognize that administration of a compound in its (R) form isequivalent, for compounds that undergo epimerization in vivo, toadministration of the compound in its (S) form.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis, high performance liquid chromatography (HPLC) and massspectrometry (MS), used by those of skill in the art to assess suchpurity, or sufficiently pure such that further purification would notdetectably alter the physical and chemical properties, such as enzymaticand biological activities, of the substance. Methods for purification ofthe compounds to produce substantially chemically pure compounds areknown to those of skill in the art. A substantially chemically purecompound may, however, be a mixture of stereoisomers. In such instances,further purification might increase the specific activity of thecompound.

As used herein, alkyl, alkenyl and alkynyl carbon chains, if notspecified, contain from 1 to 20 carbons, or 1 or 2 to 16 carbons, andare straight or branched. Alkenyl carbon chains of from 2 to 20 carbons,in certain embodiments, contain 1 to 8 double bonds and alkenyl carbonchains of 2 to 16 carbons, in certain embodiments, contain 1 to 5 doublebonds. Alkynyl carbon chains of from 2 to 20 carbons, in certainembodiments, contain 1 to 8 triple bonds, and the alkynyl carbon chainsof 2 to 16 carbons, in certain embodiments, contain 1 to 5 triple bonds.Exemplary alkyl, alkenyl and alkynyl groups herein include, but are notlimited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl,sec-butyl, tert-butyl, isopentyl, neopentyl, tert-pentyl, isohexyl,allyl (propenyl) and propargyl (propynyl). As used herein, lower alkyl,lower alkenyl, and lower alkynyl refer to carbon chains having fromabout 1 or about 2 carbons up to about 6 carbons. As used herein,“alk(en)(yn)yl” refers to an alkyl group containing at least one doublebond and at least one triple bond.

As used herein, “cycloalkyl” refers to a saturated mono- or multi-cyclicring system, in certain embodiments of 3 to 10 carbon atoms, in otherembodiments of 3 to 6 carbon atoms; cycloalkenyl and cycloalkynyl referto mono- or multicyclic ring systems that respectively include at leastone double bond and at least one triple bond. Cycloalkenyl andcycloalkynyl groups may, in certain embodiments, contain 3 to 10 carbonatoms, with cycloalkenyl groups, in further embodiments, containing 4 to7 carbon atoms and cycloalkynyl groups, in further embodiments,containing 8 to 10 carbon atoms. The ring systems of the cycloalkyl,cycloalkenyl and cycloalkynyl groups may be composed of one ring or twoor more rings which may be joined together in a fused, bridged orspiro-connected fashion. “Cycloalk(en)(yn)yl” refers to a cycloalkylgroup containing at least one double bond and at least one triple bond.

As used herein, “aryl” refers to aromatic monocyclic or multicyclicgroups containing from 6 to 19 carbon atoms. Aryl groups include, butare not limited to groups such as unsubstituted or substitutedfluorenyl, unsubstituted or substituted phenyl, and unsubstituted orsubstituted naphthyl.

As used herein, “heteroaryl” refers to a monocyclic or multicyclicaromatic ring system, in certain embodiments, of about 5 to about 15members where one or more, in one embodiment 1 to 3, of the atoms in thering system is a heteroatom, that is, an element other than carbon,including but not limited to, nitrogen, oxygen or sulfur. The heteroarylgroup may be optionally fused to a benzene ring. Heteroaryl groupsinclude, but are not limited to, furyl, imidazolyl, pyrimidinyl,tetrazolyl, thienyl, pyridyl, pyrrolyl, thiazolyl, isothiazolyl,oxazolyl, isoxazolyl, triazolyl, quinolinyl and isoquinolinyl.imidazole, triazole and pyrazole.

As used herein, “heterocyclyl” refers to a monocyclic or multicyclicnon-aromatic ring system, in one embodiment of 3 to 10 members, inanother embodiment of 4 to 7 members, in a further embodiment of 5 to 6members, where one or more, in certain embodiments, 1 to 3, of the atomsin the ring system is a heteroatom, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen or sulfur. Inembodiments where the heteroatom(s) is(are) nitrogen, the nitrogen isoptionally substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl,aralkyl, heteroaralkyl, cycloalkyl, heterocyclyl, cycloalkylalkyl,heterocyclylalkyl, acyl, guanidino, or the nitrogen may be quaternizedto form an ammonium group where the substituents are selected as above.

As used herein, “aralkyl” refers to an alkyl group in which one of thehydrogen atoms of the alkyl is replaced by an aryl group.

As used herein, “heteroaralkyl” refers to an alkyl group in which one ofthe hydrogen atoms of the alkyl is replaced by a heteroaryl group.

As used herein, “halo”, “halogen” or “halide” refers to F, Cl, Br or I.

As used herein, pseudohalides or pseudohalo groups are groups thatbehave substantially similar to halides. Such compounds can be used inthe same manner and treated in the same manner as halides. Pseudohalidesinclude, but are not limited to, cyanide, cyanate, thiocyanate,selenocyanate, trifluoromethoxy, and azide.

As used herein, “haloalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by halogen. Such groups include,but are not limited to, chloromethyl, trifluoromethyl and1-chloro-2-fluoroethyl.

As used herein, “haloalkoxy” refers to RO— in which R is a haloalkylgroup.

As used herein, “sulfinyl” or “thionyl” refers to —S(O)—. As usedherein, “sulfonyl” or “sulfuryl” refers to —S(O)₂—. As used herein,“sulfo” refers to —S(O)₂O—.

As used herein, “carboxy” refers to a divalent radical, —C(O)O—.

As used herein, “aminocarbonyl” refers to —C(O)NH₂.

As used herein, “alkylaminocarbonyl” refers to —C(O)NHR in which R isalkyl, including lower alkyl.

As used herein, “dialkylaminocarbonyl” refers to —C(O)NR′R in which R′and R are each independently alkyl, including lower alkyl; “carboxamide”refers to groups of formula —NR′COR in which R′ and R are eachindependently alkyl, including lower alkyl.

As used herein, “arylalkylaminocarbonyl” refers to —C(O)NRR′ in whichone of R and R′ is aryl, including lower aryl, such as phenyl, and theother of R and R′ is alkyl, including lower alkyl.

As used herein, “arylaminocarbonyl” refers to —C(O)NHR in which R isaryl, including lower aryl, such as phenyl.

As used herein, “hydroxycarbonyl” refers to —COOH.

As used herein, “alkoxycarbonyl” refers to —C(O)OR in which R is alkyl,including lower alkyl.

As used herein, “aryloxycarbonyl” refers to —C(O)OR in which R is aryl,including lower aryl, such as phenyl.

As used herein, “alkoxy” and “alkylthio” refer to RO— and RS—, in whichR is alkyl, including lower alkyl.

As used herein, “aryloxy” and “arylthio” refer to RO— and RS—, in whichR is aryl, including lower aryl, such as phenyl.

As used herein, “alkylene” refers to a straight, branched or cyclic, incertain embodiments straight or branched, divalent aliphatic hydrocarbongroup, in one embodiment having from 1 to about 20 carbon atoms, inanother embodiment having from 1 to 12 carbons. In a further embodimentalkylene includes lower alkylene. There may be optionally inserted alongthe alkylene group one or more oxygen, sulfur, including S(═O) andS(═O)₂ groups, or substituted or unsubstituted nitrogen atoms, including—NR— and —N⁺RR— groups, where the nitrogen substituent(s) is (are)alkyl, aryl, aralkyl, heteroaryl, heteroaralkyl or COR′, where R′ isalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, —OY or —NYY, where Y ishydrogen, alkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl. Alkylenegroups include, but are not limited to, methylene (—CH₂—), ethylene(—CH₂CH₂—), propylene (—(CH₂)₃—), methylenedioxy (—O—CH₂—O—) andethylenedioxy (—O—(CH₂)₂—O—). The term “lower alkylene” refers toalkylene groups having 1 to 6 carbons. In certain embodiments, alkylenegroups are lower alkylene, including alkylene of 1 to 3 carbon atoms.

As used herein, “azaalkylene” refers to —(CRR)_(n)—NR—(CRR)_(m)—, wheren and m are each independently an integer from 0 to 4. As used herein,“oxaalkylene” refers to —(CRR)_(n)—O—(CRR)_(m), where n and m are eachindependently an integer from 0 to 4. As used herein, “thiaalkylene”refers to —(CRR)_(n)—S—(CRR)_(m)—, —(CRR)_(n)—S(═O)—(CRR)_(m)—, and—(CRR)_(n)—S(═O)₂—(CRR)_(m)—, where n and m are each independently aninteger from 0 to 4.

As used herein, “alkenylene” refers to a straight, branched or cyclic,in one embodiment straight or branched, divalent aliphatic hydrocarbongroup, in certain embodiments having from 2 to about 20 carbon atoms andat least one double bond, in other embodiments 1 to 12 carbons. Infurther embodiments, alkenylene groups include lower alkenylene. Theremay be optionally inserted along the alkenylene group one or moreoxygen, sulfur or substituted or unsubstituted nitrogen atoms, where thenitrogen substituent is alkyl. Alkenylene groups include, but are notlimited to, —CH═CH—CH═CH— and —CH═CH—CH₂—. The term “lower alkenylene”refers to alkenylene groups having 2 to 6 carbons. In certainembodiments, alkenylene groups are lower alkenylene, includingalkenylene of 3 to 4 carbon atoms.

As used herein, “alkynylene” refers to a straight, branched or cyclic,in certain embodiments straight or branched, divalent aliphatichydrocarbon group, in one embodiment having from 2 to about 20 carbonatoms and at least one triple bond, in another embodiment 1 to 12carbons. In a further embodiment, alkynylene includes lower alkynylene.There may be optionally inserted along the alkynylene group one or moreoxygen, sulfur or substituted or unsubstituted nitrogen atoms, where thenitrogen substituent is alkyl. Alkynylene groups include, but are notlimited to, —C≡C—C≡C—, —C≡C— and —C≡C—CH₂—. The term “lower alkynylene”refers to alkynylene groups having 2 to 6 carbons. In certainembodiments, alkynylene groups are lower alkynylene, includingalkynylene of 3 to 4 carbon atoms.

As used herein, “alk(en)(yn)ylene” refers to a straight, branched orcyclic, in certain embodiments straight or branched, divalent aliphatichydrocarbon group, in one embodiment having from 2 to about 20 carbonatoms and at least one triple bond, and at least one double bond; inanother embodiment 1 to 12 carbons. In further embodiments,alk(en)(yn)ylene includes lower alk(en)(yn)ylene. There may beoptionally inserted along the alkynylene group one or more oxygen,sulfur orsubstituted or unsubstituted nitrogen atoms, where the nitrogensubstituent is alkyl. Alk(en)(yn)ylene groups include, but are notlimited to, —C═C—(CH₂)_(n)—C≡C—, where n is 1 or 2. The term “loweralk(en)(yn)ylene” refers to alk(en)(yn)ylene groups having up to 6carbons. In certain embodiments, alk(en)(yn)ylene groups have about 4carbon atoms.

As used herein, “cycloalkylene” refers to a divalent saturated mono- ormulticyclic ring system, in certain embodiments of 3 to 10 carbon atoms,in other embodiments 3 to 6 carbon atoms; cycloalkenylene andcycloalkynylene refer to divalent mono- or multicyclic ring systems thatrespectively include at least one double bond and at least one triplebond. Cycloalkenylene and cycloalkynylene groups may, in certainembodiments, contain 3 to 10 carbon atoms, with cycloalkenylene groupsin certain embodiments containing 4 to 7 carbon atoms andcycloalkynylene groups in certain embodiments containing 8 to 10 carbonatoms. The ring systems of the cycloalkylene, cycloalkenylene andcycloalkynylene groups may be composed of one ring or two or more ringswhich may be joined together in a fused, bridged or spiro-connectedfashion. “Cycloalk(en)(yn)ylene” refers to a cycloalkylene groupcontaining at least one double bond and at least one triple bond.

As used herein, “arylene” refers to a monocyclic or polycyclic, incertain embodiments monocyclic, divalent aromatic group, in oneembodiment having from 5 to about 20 carbon atoms and at least onearomatic ring, in another embodiment 5 to 12 carbons. In furtherembodiments, arylene includes lower arylene. Arylene groups include, butare not limited to, 1,2-, 1,3- and 1,4-phenylene. The term “lowerarylene” refers to arylene groups having 6 carbons.

As used herein, “heteroarylene” refers to a divalent monocyclic ormulticyclic aromatic ring system, in one embodiment of about 5 to about15 atoms in the ring(s), where one or more, in certain embodiments 1 to3, of the atoms in the ring system is a heteroatom, that is, an elementother than carbon, including but not limited to, nitrogen, oxygen orsulfur. The term “lower heteroarylene” refers to heteroarylene groupshaving 5 or 6 atoms in the ring.

As used herein, “heterocyclylene” refers to a divalent monocyclic ormulticyclic non-aromatic ring system, in certain embodiments of 3 to 10members, in one embodiment 4 to 7 members, in another embodiment 5 to 6members, where one or more, including 1 to 3, of the atoms in the ringsystem is a heteroatom, that is, an element other than carbon, includingbut not limited to, nitrogen, oxygen or sulfur.

As used herein, “substituted alkyl,” “substituted alkenyl,” “substitutedalkynyl,” “substituted cycloalkyl,” “substituted cycloalkenyl,”“substituted cycloalkynyl,” “substituted aryl,” “substitutedheteroaryl,” “substituted heterocyclyl,” “substituted alkylene,”“substituted alkenylene,” “substituted alkynylene,” “substitutedcycloalkylene,” “substituted cycloalkenylene,” “substitutedcycloalkynylene,” “substituted arylene,” “substituted heteroarylene” and“substituted heterocyclylene” refer to alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl,alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene,cycloalkynylene, arylene, heteroarylene and heterocyclylene groups,respectively, that are substituted with one or more substituents, incertain embodiments one, two, three or four substituents, where thesubstituents are as defined herein, in one embodiment selected from Q¹.

As used herein, “alkylidene” refers to a divalent group, such as ═CR′R″,which is attached to one atom of another group, forming a double bond.Alkylidene groups include, but are not limited to, methylidene (═CH₂)and ethylidene (═CHCH₃). As used herein, “arylalkylidene” refers to analkylidene group in which either R′ or R″ is an aryl group.“Cycloalkylidene” groups are those where R′ and R″ are linked to form acarbocyclic ring. “Heterocyclylidene” groups are those where at leastone of R′ and R″ contain a heteroatom in the chain, and R′ and R″ arelinked to form a heterocyclic ring.

As used herein, “amido” refers to the divalent group —C(O)NH—.“Thioamido” refers to the divalent group —C(S)NH—. “Oxyamido” refers tothe divalent group —OC(O)NH—. “Thiaamido” refers to the divalent group—SC(O)NH—. “Dithiaamido” refers to the divalent group —SC(S)NH—.“Ureido” refers to the divalent group —HNC(O)NH—. “Thioureido” refers tothe divalent group —NC(S)NH—.

As used herein, “semicarbazide” refers to —NHC(O)NHNH—. “Carbazate”refers to the divalent group —OC(O)NHNH—. “Isothiocarbazate” refers tothe divalent group —SC(O)NHNH—. “Thiocarbazate” refers to the divalentgroup —OC(S)NHNH—. “Sulfonylhydrazide” refers to the divalent group—SO₂NHNH—. “Hydrazide” refers to the divalent group —C(O)NHNH—. “Azo”refers to the divalent group —N═N—. “Hydrazinyl” refers to the divalentgroup —NH—NH—.

As used herein, “sulfonamide” refers to —RSO₂NH₂— a sulfone groupconnected to an amine group.

As used herein, “imidazole” refers to a heterocyclic aromatic organiccompound having a general formula of C₃H₄N₂.

As used herein, “triazole” refers to either one of a pair of isomericchemical compounds with molecular formula of C₂H₃N₃.

As used herein, “pyrazole” refers to a heterocyclic 5-membered ringcomposed of three carbons and two nitrogen atoms in adjacent positions.

As used herein, “adamantane” refers to a tricycloalkyl having a generalformula of C₁₀H₁₆.

Where the number of any given substituent is not specified (e.g.,haloalkyl), there may be one or more substituents present. For example,“haloalkyl” may include one or more of the same or different halogens.As another example, “C₁₋₃ alkoxyphenyl” may include one or more of thesame or different alkoxy groups containing one, two or three carbons.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem.11:942-944).

Compounds of the Invention

The compounds of this invention are:

2,3 dihydroxybenzoic acid 3,4 dihydroxyanilide (Compound 1),

3,4 dihydroxybenzoic acid 2,3 dihydroxyanilide (Compound 2),

2,3 dihydroxybenzoic acid 2,3 dihydroxyanilide (Compound 3),

3,4 dihydroxybenzoic acid 3,4 dihydroxy N-methyl anilide (Compound 4),

3,4 dihydroxybenzenesulfonic acid 3,4 dihydroxyphenylsulfonamide(Compound 5),

2,4 bis(3,4 dihydroxyphenyl)imidazole (Compound 6),

3,5 bis(3,4 dihydroxyphenyl) 1,2,4 triazole (Compound 7),

3,5 bis(3,4 dihydroxyphenyl)pyrazole (Compound 8),

1,3 bis(3,4 dihydroxyphenyl)adamantane (Compound 9),

Synthesis of the Compounds of the Invention

The compounds of this invention may be prepared by methods generallyknown to the person of ordinary skill in the art, having regard to thatknowledge and the disclosure of this application including Examples 1-5.

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as the AldrichChemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma(St. Louis, Mo.), or Lancaster Synthesis Inc. (Windham, N.H.) or areprepared by methods well known to a person of ordinary skill in the art,following procedures described in such references as Fieser and Fieser'sReagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, NewYork, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 andsupps., Elsevier Science Publishers, 1989; Organic Reactions, vols.1-40, John Wiley and Sons, New York, N.Y., 1991; March J.: AdvancedOrganic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; andLarock: Comprehensive Organic Transformations, VCH Publishers, New York,1989.

In most cases, protective groups for the hydroxy groups are introducedand finally removed. Suitable protective groups are described in Greeneet al., Protective Groups in Organic Synthesis, Second Edition, JohnWiley and Sons, New York, 1991. Other starting materials or earlyintermediates may be prepared by elaboration of the materials listedabove, for example, by methods well known to a person of ordinary skillin the art.

The starting materials, intermediates, and compounds of this inventionmay be isolated and purified using conventional techniques, includingprecipitation, filtration, distillation, crystallization,chromatography, and the like. The compounds may be characterized usingconventional methods, including physical constants and spectroscopicmethods.

Pharmacology and Utility

The compounds provided herein can be used as such, be administered inthe form of pharmaceutically acceptable salts derived from inorganic ororganic acids, or used in combination with one or more pharmaceuticallyacceptable excipients. The phrase “pharmaceutically acceptable salt”means those salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues without undue toxicity,irritation, allergic response, and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. The salts can be prepared either in situ duringthe final isolation and purification of the compounds provided herein orseparately by reacting the acidic or basic drug substance with asuitable base or acid respectively. Typical salts derived from organicor inorganic acids salts include, but are not limited to hydrochloride,hydrobromide, hydroiodide, acetate, adipate, alginate, citrate,aspartate, benzoate, bisulfate, gluconate, fumarate, hydroiodide,lactate, maleate, oxalate, palmitoate, pectinate, succinate, tartrate,phosphate, glutamate, and bicarbonate. Typical salts derived fromorganic or inorganic bases include, but are not limited to lithium,sodium, potassium, calcium, magnesium, ammonium, monoalkylammonium suchas meglumine, dialkylammonium, trialkylammonium, and tetralkylammonium.

Actual dosage levels of active ingredients and the mode ofadministration of the pharmaceutical compositions provided herein can bevaried in order to achieve the effective therapeutic response for aparticular patient. The phrase “therapeutically effective amount” of thecompound provided herein means a sufficient amount of the compound totreat disorders, at a reasonable benefit/risk ratio applicable to anymedical treatment. It will be understood, however, that the total dailyusage of the compounds and compositions of the provided will be decidedby the attending physician within the scope of sound medical judgment.The total daily dose of the compounds provided herein may range fromabout 0.1 to about 1000 mg/kg/day. For purposes of oral administration,doses can be in the range from about 1 to about 500 mg/kg/day. Ifdesired, the effective daily dose can be divided into multiple doses forpurposes of administration; consequently, single dose compositions maycontain such amounts or submultiples thereof to make up the daily dose.The specific therapeutically effective dose level for any particularpatient will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; medical history of thepatient, activity of the specific compound employed; the specificcomposition employed, age, body weight, general health, sex and diet ofthe patient, the time of administration, route of administration, theduration of the treatment, rate of excretion of the specific compoundemployed, drugs used in combination or coincidental with the specificcompound employed; and the like.

The compounds provided can be formulated together with one or morenon-toxic pharmaceutically acceptable diluents, carriers, adjuvants, andantibacterial and antifungal agents such as parabens, chlorobutanol,phenol, sorbic acid, and the like. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants. In some cases, in order to prolong theeffect of the drug, it is desirable to decrease the rate of absorptionof the drug from subcutaneous or intramuscular injection. This can beaccomplished by suspending crystalline or amorphous drug substance in avehicle having poor water solubility such as oils. The rate ofabsorption of the drug then depends upon its rate of dissolution, which,in turn, may depend upon crystal size and crystalline form. Prolongedabsorption of an injectable pharmaceutical form can be achieved by theuse of absorption delaying agents such as aluminum monostearate orgelatin.

The compound provided herein can be administered enterally orparenterally in solid or liquid forms. Compositions suitable forparenteral injection may comprise physiologically acceptable, isotonicsterile aqueous or nonaqueous solutions, dispersions, suspensions, oremulsions, and sterile powders for reconstitution into sterileinjectable solutions or dispersions. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and thelike), vegetable oils (such as olive oil), injectable organic esterssuch as ethyl oleate, and suitable mixtures thereof. These compositionscan also contain adjuvants such as preserving, wetting, emulsifying, anddispensing agents. Suspensions, in addition to the active compounds, maycontain suspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances.

The compounds provided herein can also be administered by injection orinfusion, either subcutaneously or intravenously, or intramuscularly, orintrasternally, or intranasally, or by infusion techniques in the formof sterile injectable or oleaginous suspension. The compound may be inthe form of a sterile injectable aqueous or oleaginous suspensions.These suspensions may be formulated according to the known art usingsuitable dispersing of wetting agents and suspending agents that havebeen described above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilsmay be conventionally employed including synthetic mono- ordiglycerides. In addition fatty acids such as oleic acid find use in thepreparation of injectables. Dosage regimens can be adjusted to providethe optimum therapeutic response. For example, several divided dosagesmay be administered daily or the dosage may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound may be mixed with at least one inert, pharmaceuticallyacceptable excipient or carrier, such as sodium citrate or dicalciumphosphate and/or (a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol and silicic acid; (b) binders such ascarboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose and acacia; (c) humectants such as glycerol; (d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates and sodium carbonate; (e) solutionretarding agents such as paraffin; (f) absorption accelerators such asquaternary ammonium compounds; (g) wetting agents such as cetyl alcoholand glycerol monostearate; (h) absorbents such as kaolin and bentoniteclay and (i) lubricants such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate and mixturesthereof. In the case of capsules, tablets and pills, the dosage form mayalso comprise buffering agents. Solid compositions of a similar type mayalso be employed as fillers in soft and hard-filled gelatin capsulesusing such excipients as lactose or milk sugar as well as high molecularweight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well-known in the pharmaceutical formulating art. Theymay optionally contain opacifying agents and may also be of acomposition such that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Tablets contain thecompound in admixture with non-toxic pharmaceutically acceptableexcipients that are suitable for the manufacture of tablets. Theseexcipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, maizestarch or alginic acid; binding agents, for example, maize starch,gelatin or acacia, and lubricating agents, for example, magnesiumstearate or stearic acid or tale. The tablets may be uncoated or theymay be coated by known techniques to delay disintegration and absorptionin the gastrointestinal tract and thereby provide a sustained actionover a longer period. For example, a time delay material such asglycerol monostearate or glycerol distearate may be employed.Formulations for oral use may also be presented as hard gelatin capsuleswherein the compound is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate or kaolin, or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, peanut oil, liquid paraffin or olive oil.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan andmixtures thereof. Besides inert diluents, the oral compositions may alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring and perfuming agents.

Aqueous suspensions contain the compound in admixture with excipientssuitable for the manufacture of aqueous suspensions. Such excipients aresuspending agents, for example, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethyl cellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents may be naturally occurring phosphatides, for examplelecithin, or condensation products of an alkylene oxide with fattyacids, for example polyoxyethylene stearate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for example,heptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids such as hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters from fatty acids and a hexitolanhydrides, for example, polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for example,ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents, or one or more sweetening agents, such as sucroseor saccharin.

Oily suspensions may be formulated by suspending the compound in avegetable oil, for example arachis oil, olive oil, sesame oil, orcoconut oil or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents, such as those set forthbelow, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid. Dispersible powders and granulessuitable for preparation of an aqueous suspension by the addition ofwater provide the active ingredient in admixture with a dispersing orwetting agent, a suspending agent and one or more preservatives.Suitable dispersing or wetting agents and suspending agents areexemplified by those already described above. Additional excipients, forexample sweetening, flavoring and agents, may also be present.

The compounds provided herein may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil, for example olive oilor arachis oils, or a mineral oil, for example liquid paraffin ormixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally occurring phosphatides, for example soy bean, lecithin, andoccurring phosphatides, for example soy bean, lecithin, and esters orpartial esters derived from fatty acids and hexitol anhydrides, forexample sorbitan monooleate, and condensation products of the saidpartial esters with ethylene oxide, for example polyoxyethylene sorbitanmonooleate. The emulsion may also contain sweetening and flavoringagents. Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, sorbitol or sucrose. Such formulations may alsocontain a demulcent, a preservative and flavoring and coloring agents.

In one embodiment, the compounds are formulated in dosage unit form forease of administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for the subjects to be treated; each containing atherapeutically effective quantity of the compound and at least onepharmaceutical excipient. A drug product will comprise a dosage unitform within a container that is labeled or accompanied by a labelindicating the intended method of treatment, such as the treatment of anβ-amyloid disease, for example an amyloidosis such as Alzheimer'sdisease or a disease associated with α-synuclein fibril formation suchas Parkinson's disease. Compositions for rectal or vaginaladministration are preferably suppositories which can be prepared bymixing the compounds provided herein with suitable non-irritatingexcipients or carriers such as cocoa butter, polyethylene glycol or asuppository wax which are solid at room temperature but liquid at bodytemperature and therefore melt in the rectum or vaginal cavity andrelease the active compound.

Compounds provided herein can also be administered in the form ofliposomes. Methods to form liposomes are known in the art (Prescott,Ed., Methods in Cell Biology 1976, Volume XIV, Academic Press, New York,N.Y.) As is known in the art, liposomes are generally derived fromphospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals which are dispersed in anaqueous medium. Any non-toxic, physiologically acceptable andmetabolizable lipid capable of forming liposomes can be used. Thepresent compositions in liposome form can contain, in addition to acompound provided herein, stabilizers, preservatives, excipients and thelike. The preferred lipids are natural and synthetic phospholipids andphosphatidyl cholines (lecithins).

The compounds provided herein can also be administered in the form of a‘prodrug’ wherein the active pharmaceutical ingredients, are released invivo upon contact with hydrolytic enzymes such as esterases andphophatases in the body. The term “pharmaceutically acceptable prodrugs”as used herein represents those prodrugs of the compounds providedherein, which are, within the scope of sound medical judgment, suitablefor use in contact with the tissues without undue toxicity, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio, and effective for their intended use. A thoroughdiscussion is provided in T. Higuchi and V. Stella (Higuchi, T. andStella, V. Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S.Symposium Series; Edward B. Roche, Ed., Bioreversible Carriers in DrugDesign 1987, American Pharmaceutical Association and Pergamon Press),which is incorporated herein by reference.

The compounds provided herein, or pharmaceutically acceptablederivatives thereof, may also be formulated to be targeted to aparticular tissue, receptor, or other area of the body of the subject tobe treated. Many such targeting methods are well known to those of skillin the art. All such targeting methods are contemplated herein for usein the instant compositions. For non-limiting examples of targetingmethods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359,6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082,6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252,5,840,674, 5,759,542 and 5,709,874.

In one embodiment, liposomal suspensions, including tissue-targetedliposomes, such as tumor-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art. For example, liposomeformulations may be prepared as described in U.S. Pat. No. 4,522,811.Briefly, liposomes such as multilamellar vesicles (MLV's) may be formedby drying down egg phosphatidyl choline and brain phosphatidyl serine(7:3 molar ratio) on the inside of a flask. A solution of a compoundprovided herein in phosphate buffered saline lacking divalent cations(PBS) is added and the flask shaken until the lipid film is dispersed.The resulting vesicles are washed to remove unencapsulated compound,pelleted by centrifugation, and then resuspended in PBS.

Sustained Release Formulations

The invention also includes the use of sustained release formulations todeliver the compounds of the present invention to the desired target(i.e. brain or systemic organs) at high circulating levels (between 10⁻⁹and 10⁻⁴ M) are also disclosed. In a preferred embodiment for thetreatment of Alzheimer's or Parkinson's disease, the circulating levelsof the compounds is maintained up to 10⁻⁷ M. The levels are eithercirculating in the patient systemically, or in a preferred embodiment,present in brain tissue, and in a most preferred embodiments, localizedto the β-amyloid or α-synuclein fibril deposits in brain or othertissues.

It is understood that the compound levels are maintained over a certainperiod of time as is desired and can be easily determined by one skilledin the art using this disclosure and compounds of the invention. In apreferred embodiment, the invention includes a unique feature ofadministration comprising a sustained release formulation so that aconstant level of therapeutic compound is maintained between 10⁻⁸ and10⁻⁶ M between 48 to 96 hours in the sera.

Such sustained and/or timed release formulations may be made bysustained release means of delivery devices that are well known to thoseof ordinary skill in the art, such as those described in U.S. Pat. Nos.3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384;5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;5,354,556 and 5,733,566, the disclosures of which are each incorporatedherein by reference. These pharmaceutical compositions can be used toprovide slow or sustained release of one or more of the active compoundsusing, for example, hydroxypropylmethyl cellulose, other polymermatrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, liposomes, microspheres, or the like. Suitablesustained release formulations known to those skilled in the art,including those described herein, may be readily selected for use withthe pharmaceutical compositions of the invention. Thus, single unitdosage forms suitable for oral administration, such as, but not limitedto, tablets, capsules, gelcaps, caplets, powders and the like, that areadapted for sustained release are encompassed by the present invention.

In a preferred embodiment, the sustained release formulation containsactive compound such as, but not limited to, microcrystalline cellulose,maltodextrin, ethylcellulose, and magnesium stearate. As describedabove, all known methods for encapsulation which are compatible withproperties of the disclosed compounds are encompassed by this invention.The sustained release formulation is encapsulated by coating particlesor granules of the pharmaceutical composition of the invention withvarying thickness of slowly soluble polymers or by microencapsulation.In a preferred embodiment, the sustained release formulation isencapsulated with a coating material of varying thickness (e.g. about 1micron to 200 microns) that allow the dissolution of the pharmaceuticalcomposition about 48 hours to about 72 hours after administration to amammal. In another embodiment, the coating material is a food-approvedadditive.

In another embodiment, the sustained release formulation is a matrixdissolution device that is prepared by compressing the drug with aslowly soluble polymer carrier into a tablet. In one preferredembodiment, the coated particles have a size range between about 0.1 toabout 300 microns, as disclosed in U.S. Pat. Nos. 4,710,384 and5,354,556, which are incorporated herein by reference in theirentireties. Each of the particles is in the form of a micromatrix, withthe active ingredient uniformly distributed throughout the polymer.

Sustained release formulations such as those described in U.S. Pat. No.4,710,384, which is incorporated herein by reference in its entirety,having a relatively high percentage of plasticizer in the coating inorder to permit sufficient flexibility to prevent substantial breakageduring compression are disclosed. The specific amount of plasticizervaries depending on the nature of the coating and the particularplasticizer used. The amount may be readily determined empirically bytesting the release characteristics of the tablets formed. If themedicament is released too quickly, then more plasticizer is used.Release characteristics are also a function of the thickness of thecoating. When substantial amounts of plasticizer are used, the sustainedrelease capacity of the coating diminishes. Thus, the thickness of thecoating may be increased slightly to make up for an increase in theamount of plasticizer. Generally, the plasticizer in such an embodimentwill be present in an amount of about 15 to 30% of the sustained releasematerial in the coating, preferably 20 to 25%, and the amount of coatingwill be from 10 to 25% of the weight of the active material, preferably15 to 20%. Any conventional pharmaceutically acceptable plasticizer maybe incorporated into the coating.

The compounds of the invention can be formulated as a sustained and/ortimed release formulation. All sustained release pharmaceutical productshave a common goal of improving drug therapy over that achieved by theirnon-sustained counterparts. Ideally, the use of an optimally designedsustained release preparation in medical treatment is characterized by aminimum of drug substance being employed to cure or control thecondition. Advantages of sustained release formulations may include: 1)extended activity of the composition, 2) reduced dosage frequency, and3) increased patient compliance. In addition, sustained releaseformulations can be used to affect the time of onset of action or othercharacteristics, such as blood levels of the composition, and thus canaffect the occurrence of side effects.

The sustained release formulations of the invention are designed toinitially release an amount of the therapeutic composition that promptlyproduces the desired therapeutic effect, and gradually and continuallyrelease of other amounts of compositions to maintain this level oftherapeutic effect over an extended period of time. In order to maintainthis constant level in the body, the therapeutic composition must bereleased from the dosage form at a rate that will replace thecomposition being metabolized and excreted from the body.

The sustained release of an active ingredient may be stimulated byvarious inducers, for example pH, temperature, enzymes, water, or otherphysiological conditions or compounds. The term “sustained releasecomponent” in the context of the present invention is defined herein asa compound or compounds, including, but not limited to, polymers,polymer matrices, gels, permeable membranes, liposomes, microspheres, orthe like, or a combination thereof, that facilitates the sustainedrelease of the active ingredient.

If the complex is water-soluble, it may be formulated in an appropriatebuffer, for example, phosphate buffered saline, or other physiologicallycompatible solutions. Alternatively, if the resulting complex has poorsolubility in aqueous solvents, then it may be formulated with anon-ionic surfactant such as Tween, or polyethylene glycol. Thus, thecompounds and their physiologically solvents may be formulated foradministration by inhalation or insufflation (either through the mouthor the nose) or oral, buccal, parenteral, or rectal administration, asexamples.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. In a preferred embodiment,the compounds of the present invention are formulated as controlledrelease powders of discrete microparticles that can be readilyformulated in liquid form. The sustained release powder comprisesparticles containing an active ingredient and optionally, an excipientwith at least one non-toxic polymer.

The powder can be dispersed or suspended in a liquid vehicle and willmaintain its sustained release characteristics for a useful period oftime. These dispersions or suspensions have both chemical stability andstability in terms of dissolution rate. The powder may contain anexcipient comprising a polymer, which may be soluble, insoluble,permeable, impermeable, or biodegradable. The polymers may be polymersor copolymers. The polymer may be a natural or synthetic polymer.Natural polymers include polypeptides (e.g., zein), polysaccharides(e.g., cellulose), and alginic acid. Representative synthetic polymersinclude those described, but not limited to, those described in column3, lines 33-45 of U.S. Pat. No. 5,354,556, which is incorporated byreference in its entirety. Particularly suitable polymers include thosedescribed, but not limited to those described in column 3, line46-column 4, line 8 of U.S. Pat. No. 5,354,556 which is incorporated byreference in its entirety.

The sustained release compounds of the invention may be formulated forparenteral administration, e.g., by intramuscular injections or implantsfor subcutaneous tissues and various body cavities and transdermaldevices. In one embodiment, intramuscular injections are formulated asaqueous or oil suspensions. In an aqueous suspension, the sustainedrelease effect is due to, in part, a reduction in solubility of theactive compound upon complexation or a decrease in dissolution rate. Asimilar approach is taken with oil suspensions and solutions, whereinthe release rate of an active compound is determined by partitioning ofthe active compound out of the oil into the surrounding aqueous medium.Only active compounds which are oil soluble and have the desiredpartition characteristics are suitable. Oils that may be used forintramuscular injection include, but are not limited to, sesame, olive,arachis, maize, almond, soybean, cottonseed and castor oil.

A highly developed form of drug delivery that imparts sustained releaseover periods of time ranging from days to years is to implant adrug-bearing polymeric device subcutaneously or in various bodycavities. The polymer material used in an implant, which must bebiocompatible and nontoxic, include but are not limited to hydrogels,silicones, polyethylenes, ethylene-vinyl acetate copolymers, orbiodegradable polymers.

Evaluation of the Activity of the Compounds

The biological activity of the compounds provided herein asdisruptors/inhibitors of Alzheimer's disease β-amyloid protein (Aβ)fibrils, and Parkinson's disease α-synuclein fibrils was assessed bydetermining the efficacy of the compounds to cause adisassembly/disruption of pre-formed amyloid fibrils of Alzheimer'sdisease (i.e. consisting of Aβ 1-42 fibrils), and Parkinson's diseaseα-synuclein fibrils. In one study, Thioflavin T fluorometry was used todetermine the effects of the compounds, and of EDTA (as a negativecontrol). In this assay Thioflavin T binds specifically to fibrillaramyloid, and this binding produces a fluorescence enhancement at 485 nmthat is directly proportional to the amount of fibrils present. Thehigher the fluorescence, the greater the amount of fibrils present (Nakiet al, Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci.2:404-410, 1993; Amyloid: Int. J. Exp. Clin. Invest. 2:1-6, 1995).

In the Congo red binding assay the ability of a given test compound toalter amyloid (Aβ1-42 fibrils, or α-synuclein fibrils) binding to Congored was quantified. In this assay, Aβ 1-42 fibrils, or α-synucleinfibrils and test compounds were incubated for 3 days and then vacuumfiltered through a 0.2 μm filter. The amount of Aβ 1-42 fibrils, orα-synuclein fibrils retained in the filter was then quantitatedfollowing staining of the filter with Congo red. After appropriatewashing of the filter, any lowering of the Congo red color on the filterin the presence of the test compound (compared to the Congo red stainingof the amyloid protein in the absence of the test compound) wasindicative of the test compound's ability to diminish/alter the amountof aggregated and congophilic Aβ 1-42 fibrils, or α-synuclein fibrils.

Combination Therapy

In another embodiment, the compounds may be administered in combination,or sequentially, with another therapeutic agent. Such other therapeuticagents include those known for treatment, prevention, or amelioration ofone or more symptoms of amyloidosis and synuclein diseases. Suchtherapeutic agents include, but are not limited to, donepezilhydrochloride (Aracept), rivastigmine tartrate (Exelon), tacrinehydrochloride (Cognex) and galantamine hydrobromide (Reminyl).

Methods of Use of the Compounds and Compositions

The compounds and compositions provided herein are useful in methods oftreatment, prevention, or amelioration of one or more symptoms ofβ-amyloid diseases or disorders, including but not limited to diseasesassociated with the formation, deposition, accumulation, or persistenceof β-amyloid fibrils. In certain embodiments, the compounds andcompositions provided herein are used for treatment, prevention, oramelioration of one or more symptoms of diseases including, but notlimited to of Alzheimer's disease. Down's syndrome, hereditary cerebralhemorrhage with amyloidosis of the Dutch type, and cerebral β-amyloidangiopathy.

Also provided are methods to inhibit or prevent α-synuclein fibrilformation, methods to inhibit or prevent α-synuclein fibril growth, andmethods to cause disassembly, disruption, and/or disaggregation ofpreformed α-synuclein fibrils and α-synuclein-associated proteindeposits.

In certain embodiments, the synuclein diseases or synucleinopathiestreated, prevented or whose symptoms are ameliorated by the compoundsand compositions provided herein include, but are not limited todiseases associated with the formation, deposition, accumulation, orpersistence of synuclein fibrils, including α-synuclein fibrnls. Incertain embodiments, such diseases include Parkinson's disease, familialParkinson's disease, Lewy body disease, the Lewy body variant ofAlzheimer's disease, dementia with Lewy bodies, multiple system atrophy,and the Parkinsonism-dementia complex of Guam.

The following non-limiting Examples are given by way of illustrationonly and are not considered a limitation of this invention, manyapparent variations of which are possible without departing from thespirit or scope thereof.

EXAMPLES General Experimental Procedures

All solvents were distilled before use and were removed by rotaryevaporation at temperatures up to 35° C. Merck silica gel 60, 200-400mesh, 40-63 μm, was used for silica gel flash chromatography. TLC wascarried out using Merck DC-plastikfolien Kieselgel 60 F254, firstvisualised with a UV lamp, and then by dipping in a vanillin solution(1% vanillin, 1% H₂SO₄ in EtOH), and heating. Mass spectra were recordedon a Kratos MS-80 instrument. NMR spectra, at 25° C., were recorded at500 or 300 MHz for ¹H and 125 or 75 MHz for ¹³C on Varian INOVA-500 orVXR-300 spectrometers. Chemical shifts are given in ppm on the δ scalereferenced to the solvent peaks: CHCl₃ at 7.25 and CDCl₃ at 77.0 ppm or(CH₃)₂CO at 2.15 and (CD₃)₂CO at 30.5 ppm or CH₃OD at 3.30 and CD₃OD at39.0 ppm.

HPLC Conditions

Samples were analysed using an Agilent HP1100 instrument, operated withEzChrom Elite software, and fitted with a C18 column (Phenomenex Prodigy5 μm 100 A, 250×4.6 mm) with a guard column (Phenomenex ODS 4×3 mm, 5μm) held at 30° C. Peaks were detected at 280 nm. The mobile phase wasacetonitrile in water (with 0.1% TFA): t₀=11%, t₂₀=11%, t₃₀=100%,t₃₁=11%, t₄₀=11%. The flow rate was 1 mL/min and the injection volume of5 μL.

Example 1 Synthesis of Sulfonamide 2 3,4 dihydroxybenzenesulfonic acid3,4 dihydroxyphenylsulfonamide (Compound 5)

Synthesis of the sulfonamide 2 was accomplished by reaction of3,4-methylenedioxybenzenesulfonyl chloride (prepared from1,2-methylenedioxybenzene (Tao, E. V. P.; Miller, W. D. U.S. Pat. No.5,387,681. 1995)) with 3,4-methylenedioxyaniline to give the sulfonamide1 in good yield. Deprotection with boron tribromide under standardconditions gave the free phenolic sulfonamide in reasonable yield.

To a stirred solution of 1,3-benzodioxole-5-sulfonyl chloride (Tao, E.V. P.; Miller, W. D. U.S. Pat. No. 5,387,681. 1995) (1 g) indichloromethane (DCM) (10 ml) was added a solution of3,4-methylenedioxyaniline (0.62 g) in dichloromethane (10 ml) followedby pyridine (1 ml). The mixture was refluxed for 2 hours, cooled,diluted with dichloromethane (150 ml), washed with aqueous HCl (1M,2×100 ml), dried, then evaporated in vacuo to give the crude product asa brown gum. Purification by column chromatography over silica geleluting with 5-10% ethyl acetate in dichloromethane gave the puresulphonamide 1 as a pale brown gum (1.34 g, 92%). Crystallisation from95% ethanol gave the product as pale brown crystals.

HPLC 29.6 minutes.

¹H NMR ((CD₃)₂CO) 8.75 (1H, s), 7.39 (2H, dd, J 2, 9 Hz), 7.24 (1H, d, J2 Hz), 7.02 (1H, d, J 9 Hz), 6.86 (1H, d, J 2 Hz), 6.81 (1H, d, J 9 Hz),6.72 (2H, dd, J 2, 9 Hz), 6.23 (2H, s) and 6.06 (2H, s).

HREIMS Found, 344.0201; MNa⁺, C₁₄H₁₁NNaO₆S requires 344.0199.

To a solution of the sulphonamide 1 (0.7 g) in dry DCM (50 ml) was addedboron tribromide (0.5 ml) and the mixture left at room temperature for 3hours. Methanol (dropwise then 5 ml) was added carefully then thereaction left at room temperature for 24 hours. The mixture wasevaporated in vacuo to 1 ml, then more methanol (20 ml) was added, thiswas repeated four times, then the solvents were removed by evaporationin vacuo.

Purification by column chromatography over silica gel eluting with 0-20%methanol in chloroform gave the product as a pale brown gum. Furtherpurification over C-18 reverse phase silica eluting with 0-50%acetonitrile in water, followed by freeze drying, gave the pure product2 as a light brown powder (295 mg, 45%).

HPLC 12.9 minutes 95%

¹H NMR (CD₃OD) 7.05 (1H, d, J 2 Hz), 7.03 (2H, dd, J 2, 9 Hz), 6.76 (1H,d, J 9 Hz), 6.57 (1H, d, J 2 Hz), 6.56 (1H, d, J 9 Hz) and 6.31 (2H, dd,J 2, 9 Hz).

HREIMS Found, 296.0241, M⁻, C₁₂H₁₀NO₆S requires, 296.0234.

Example 2 Synthesis of Imidazole 4 2,4 bis(3,4 dihydroxyphenyl)imidazole(Compound 6)

The imidazole ring was formed according to the method described by Li etal. (Li et al. Organic Process Research and Development 2002, 6, 682-3)from the amidinobenzene, formed from piperonylonitrile (Thurkauf et al.J Med. Chem. 1995, 38 (12), 2251-2255) and the bromoketone (Castedo etal. Tetrahedron 1982, 38 (11), 1569-70) formed from3,4-methylenedioxyacetophenone according to the method described by Leeet al. (Korean Chem. Soc. 2003, 24 (4), 407-408). Deprotection withboron tribromide under standard conditions gave the free phenolicimidazole in good yield.

According to the process described by Li, a mixture of 3-amidinobenzene(Thurkauf et al. J Med. Chem. 1995, 38 (12), 2251-2255) (0.5 g, 3 mmol)and potassium bicarbonate (1.20 g, 12 mmol) in tetrahydrofuran (THF) (16ml) and water (4 ml) was heated vigorously at reflux. Bromoketone(Castedo et al. Tetrahedron 1982, 38 (11), 1569-70; and Lee et al.Korean Chem. Soc. 2003, 24 (4), 407-408) (0.729 g, 3 mmol) in THF (4 ml)was added over 30 minutes and reflux was maintained for a further 2hours. The THF was then removed by evaporation in vacuo and the residueextracted into ethyl acetate, dried and evaporated in vacuo to give thecrude product as a brown solid. Crystallisation from 95% ethanol gavethe pure imidazole 3 as a pale yellow crystalline solid (0.54 g, 58%).

HPLC 27.9 minutes. NMR ((CD₃)₂CO) 7.45-7.70 (5H, m), 7.02 (1H, d, J 9Hz), 6.95 (1H, d, J 9 Hz), 6.15 (2H, s) and 6.09

(2H, s) HREIMS Found, 309.0875; MH⁺, C₁₇H₁₂N₂O₄ requires, 309.0870.

To a solution of the imidazole 3 (0.5 g) in dry DCM (50 ml) was addedboron tribromide (1.0 ml) and the mixture left at room temperature for 3hours. Methanol (dropwise then 5 ml) was added carefully then thereaction left at room temperature for 24 hours. The mixture wasevaporated in vacuo to 1 ml, then more methanol (30 ml) was added, thiswas repeated four times, then the solvents were removed by evaporationin vacuo.

Purification by column chromatography over silica gel eluting with 0-20%methanol in chloroform gave the product 4 as a pale brown solid (0.27 g,58%).

HPLC 16.3 minutes 99%

¹H NMR (CD₃OD) 7.59 (1H, s), 7.36 (1H, d, J 2 Hz), 7.31 (2H, dd, J 2, 9Hz), 7.16 (1H, d, J 2 Hz), 7.10 (2H, dd, J 2, 9 Hz), 6.98 (1H, d, J 9Hz) and 6.88 (1H, d, J 9 Hz).

HREIMS Found, 285.0873; MH⁺, C₁₅H₁₃N₂O₄ requires 285.0870.

Example 3 Synthesis of Triazole 7 3,5 bis(3,4 dihydroxyphenyl) 1,2,4triazole (Compound 7)

The 4-aminotriazole ring was formed by a dimerization reaction ofpiperonylonitrile according to the method described by Bentiss (Bentisset al. J Heterocyclic Chem. 1999, 36, 149-152) and then deamination wascarried out according to the method described by Bentiss (Bentiss et al.J. Heterocyclic Chem. 2002, 39, 93-96.) to give the triazole 6 in goodyield. Deprotection with boron tribromide under standard conditions gavethe free phenolic triazole 7 in good yield.

According to the process described by Bentiss (Bentiss et al. JHeterocyclic Chem. 1999, 36, 149-152) a mixture of aromatic nitrile (1g), hydrazine hydrate (1 g) and hydrazine hydrochloride (0.5 g) insolution in ethylene glycol (5 ml) was heated to 130° C. for 5 hours.The solution was cooled then diluted with water (7 ml), the solidproduct was filtered, washed with DCM then dried to give the crudeproduct. Recrystalisation from methanol gave the pure 4-aminotriazole 5,as a pale yellow solid (0.65 g, 66%).

HPLC 27.0 minutes.

¹H NMR ((CD₃)₂CO) 7.62 (2H, dd, J 2, 9 Hz), 7.42 (2H, d, J 2 Hz), 6.94(2H, d, J 9 Hz), 6.15 (2H, s) and 5.93 (4H, s).

HREIMS Found, 325.0937; MH⁺, C₁₆H₁₃N₄O₄ requires 325.0931.

According to the process described by Bentiss (Bentiss et al. J.Heterocyclic Chem. 2002, 39, 93-96) to a stirred solution of aminotriazole 5 (0.5 g) in an aqueous solution of hypophosphorus acid (50%, 5ml) a solution of sodium nitrite (0.6 g) in water (1.5 ml) was addedslowly. The mixture was stirred at room temperature for a further hourthen the pale orange precipitate was collected, washed with water anddried to give the triazole 6 (0.38, 80%).

HPLC 29.48 minutes.

¹H NMR ((CD₃)₂CO) 7.81 (2H, dd, J 2, 9 Hz), 7.70 (2H, d, J 2 Hz), 7.10(2H, d, J 9 Hz) and 6.20 (4H, s). HREIMS Found, 310.0818; C₁₆H₁₂N₃O₄requires 310.0822.

To a solution of the triazole 6 (0.5 g) in dry DCM (50 ml) was addedboron tribromide (1.0 ml) and the mixture left at room temperature for 3hours. Methanol (dropwise then 5 ml) was added carefully then thereaction left at room temperature for 24 h. The mixture was evaporatedin vacuo to 1 ml, then more methanol (30 ml) was added, this wasrepeated four times, then the solvents were removed by evaporation invacuo.

Purification by column chromatography over silica gel eluting with 0-20%methanol in chloroform gave the product 7 as a pale brown solid (0.24 g,52%).

HPLC 16.1 minutes 97%

¹H NMR (CD₃OD) 7.46 (2H, d, J 2 Hz), 7.41 (2H, dd, J 2, 9 Hz), 7.15 (1H,s) and 6.96 (2H, d, J 9 Hz).

HREIMS Found, 286.0815; MH⁺, C₁₄H₁₂N₃O₄ requires 286.0822.

Example 4 Synthesis of Pyrazole 9 3,5 bis(3,4 dihydroxyphenyl)pyrazole(Compound 8)

Reaction of the 1,3-diketone (Lopez et al. Planta Med. 1998, 64 (1),76-77) (prepared according to the method described by Choshi et al.(Chem. Pharm. Bull. 1992, 40 (4), 1047-1049) with hydrazine hydrateaccording to the method described by Fink et al. (Chemistry and Biology1999, 6, 205-219) gave the pyrazole 8 in good yield. Deprotection withboron tribromide under standard conditions gave the free phenolicpyrazole 9 in good yield.

According to the method described by Fink et al. (Chemistry and Biology1999, 6, 205-219) a suspension of the diketone (Choshi et al. Chem.Pharm. Bull. 1992, 40 (4), 1047-1049 and Lopez et al. Planta Med. 1998,64 (1), 76-77) (1 g) and hydrazine HCl (1 g, 5 equivs) in DMF/THF (3:1,12 ml) was heated to reflux for 24 h. Water was added and the mixtureextracted into dichloromethane, dried and evaporated in vacuo to givethe crude product 8 as a yellow solid. Purification by columnchromatography over silica gel eluting with 0-20% ethyl acetate indichloromethane gave the pyrazole 8 as a pale yellow solid (0.49 g,50%).

HPLC 30.3 minutes

¹H NMR ((CD₃)₂CO) 7.47 (2H, dd, J 2, 9 Hz), 7.46 (2H, d, J 2 Hz), 7.04(1H, s), 7.02 (2H, d, J 9 Hz) and 6.14 (4H, s).

HREIMS Found, 309.0859; MH⁺, C₁₇H13N2O4 requires 309.0870.

To a solution of the pyrazole 8 (0.46 g) in dry DCM (50 ml) was addedboron tribromide (0.4 ml) and the mixture left at room temperature for 3hours. Methanol (dropwise then 5 ml) was added carefully then thereaction left at room temperature for 24 hours. The mixture wasevaporated in vacuo to 1 ml, then more methanol (30 ml) was added, thiswas repeated four times, then the solvents were removed by evaporationin vacuo.

Purification by column chromatography over silica gel eluting with 0-20%methanol in chloroform gave the pyrazole 9 as a pale yellow solid.(0.285 g, 67%).

HPLC 25.9 minutes 98%

¹H NMR (CD₃OD) 7.26 (2H, d, J 2 Hz), 7.22 (2H, dd, J 2, 9 Hz), 7.15 (1H,s) and 6.93 (2H, d, J 9 Hz).

HREIMS Found, 285.0879; C₁₅H₁₃N₂O₄ requires, 285.0870.

Example 5 Synthesis of Adamantane 10 1,3 bis(3,4dihydroxyphenyl)adamantane (Compound 9)

Reaction of catechol with 1,3-adamantane-diol according to the methoddescribed by Lu et al (Lu et al. J Med Chem 2005, 48 (14), 4576-4585)gave the adduct 10 in reasonable yield.

According to the method described by Lu a solution of catechol (1.0 g)and adamantane diol (0.5 g) in methanesulfonic acid (2 ml) was heated to80° C. for 3 hours, then left at room temperature overnight. Water wasadded and the mixture extracted into 10% methanol in chloroform whichwas dried and evaporated in vacuo to give a white solid. Purification bycolumn chromatography over silica gel eluting with 0-20% methanol inchloroform gave the product as a white solid. Crystallisation fromdiethyl ether/40% petroleum ether then gave the pure product 10 as awhite crystalline solid (210 mg, 20%).

HPLC 29.8 minutes 98%

¹H NMR (CD₃OD) 6.82 (2H, t, J 1.5 Hz), 6.68 (4H, d, J 1.5 Hz), 2.22 (2H,bs), 1.87 (8H, m) and 1.77 (2H, bs).

HREIMS Found, 387.1369; MCl⁻, C₂₂H₂₄ClO₄ requires, 387.1369.

Example 6 Compounds of this Invention are Potent Disrupters ofAlzheimer's Aβ 1-42 Fibrils

The compounds prepared in the preceding Examples were found to be potentdisruptors/inhibitors of Alzheimer's disease β-amyloid protein (Aβ)fibrnls. In a set of studies, the efficacy of the compounds to cause adisassembly/disruption of pre-formed amyloid fibrils of Alzheimer'sdisease (i.e. consisting of Aβ 1-42 fibrils) was analyzed.

Part A—Thioflavin T fluorometry

In one study, Thioflavin T fluorometry was used to determine the effectsof the compounds, and of EDTA (as a negative control). In this assayThioflavin T binds specifically to fibrillar amyloid, and this bindingproduces a fluorescence enhancement at 485 nm that is directlyproportional to the amount of amyloid fibrils formed. The higher thefluorescence, the greater the amount of amyloid fibrils formed (Naki etal., Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci. 2:404-410,1993; Amyloid: Int. J. Exp. Clin. Invest. 2:1-6, 1995).

In this study, 30 μL of a 1 mg/mL solution (in distilled water) ofpre-fibrillized Aβ 1-42 (rPeptide) was incubated at 37° C. for 3 dayseither alone, or in the presence of one of the compounds or EDTA (atAβ:test compound weight ratios of 1:1, 1:0.1, 1:0.01 or 1:0.001).Following 3-days of co-incubation, 50 μl of each incubation mixture wastransferred into a 96-well microtiter plate containing 150 μl ofdistilled water and 50 μl of a Thioflavin T solution (i.e. 500 mMThioflavin T in 250 mM phosphate buffer, pH 6.8). The emissionfluorescence was read at 485 nm (444 nm excitation wavelength) using anELISA plate fluorometer after subtraction with buffer alone or compoundalone, as blank.

The results of the 3-day incubations are illustrated graphically in FIG.5. For example, whereas EDTA (‘−C’ in FIG. 5) caused no significantinhibition of Aβ 1-42 fibrils at all concentrations tested, thecompounds all caused a dose-dependent disruption/disassembly ofpreformed Aβ 1-42 fibrils. All of the compounds tested were effective indisrupting pre-formed Aβ 1-42 fibrils similar to the results obtainedfrom a positive control compound (‘+C’ in FIG. 5). For example, all ofthe compounds caused at least 96% inhibition when used at an Aβ:testcompound wt/wt ratio of 1:1 compared to 99% for the control. At anAβ:test compound wt/wt ratio of 1:0.1 the levels of inhibition rangedfrom 86 to 95% compared to 92% for the control. This study indicatedthat the compounds of this invention are potent disruptors/inhibitors ofAlzheimer's disease type Aβ fibrils, and usually exert their effects ina dose-dependent manner.

Part B: Congo Red

In the Congo red binding assay the ability of a test compound to alterβ-amyloid binding to Congo red is quantified. In this assay, Aβ 1-42 (asprepared for the Thio T assay) and test compounds were incubated for 3days and then vacuum filtered through a 0.2 μm filter. The amount of Aβ1-42 retained in the filter was then quantitated following staining ofthe filter with Congo red. After appropriate washing of the filter, anylowering of the Congo red color on the filter in the presence of thetest compound (compared to the Congo red staining of the amyloid proteinin the absence of the test compound) was indicative of the testcompound's ability to diminish/alter the amount of aggregated andcongophilic Aβ.

In one study, the ability of Aβ fibrils to bind Congo red in the absenceor presence of increasing amounts of the compounds or EDTA (at Aβ:testcompound weight ratios of 1:1, 1:0.1, 1:0.01 or 1:0.001) was determined.The results of 3-day incubations are illustrated graphically in FIG. 6.Whereas EDTA (‘−C’ in FIG. 6) caused no significant inhibition of Aβ1-42 fibril binding to Congo red at all concentrations tested, thecompounds caused a dose-dependent inhibition of Aβ binding to Congo red,some exceeding the effects of the positive control compound (‘+C’ inFIG. 6). For example, the positive control compound caused a significant(p<0.01) 73.5% inhibition of Congo red binding to Aβ 1-42 fibrils whenused at an Aβ:test compound wt/wt ratio of 1:1, and a significant(p<0.01) 10.4% inhibition of Congo red binding when used at an Aβ:testcompound wt/wt ratio of 1:0.1. Compounds 6, 8 and 9 exceed the resultsof the positive control compound at both the above noted ratios. Similarto the results for Thio T assay, this study also indicated thatcompounds of this invention are potent inhibitors of Aβ fibril bindingto Congo red, and usually exert their effects in a dose-dependentmanner.

Part D—Circular Dichroism Spectroscopy Data

Circular dichroism (CD) spectroscopy is a method that can be used todetermine the effects of test compounds on disruption of the secondarystructural conformation of amyloid fibrils. In one study, as describedin this example, circular dichroism spectroscopy was used to determinethe effects of different compounds of the invention on the β-sheetconformation of Aβ₁₋₄₂ fibrils. For this study, Aβ₁₋₄₂ (rPeptide Inc.,Bogart, Ga.) was first lyophilized from a 50 mM NaOH solution, the pHbeing maintained above 10 prior to freezing and lyophilization. Thepeptide was then reconstituted in 20 mM acetate buffer, pH 4.0, at aconcentration of 1 mg/ml. Dilution and addition of test compounds orvehicle was performed such that the final concentration of peptide was0.5 mg/ml and the Aβ₁₋₄₂:test compound wt/wt ratios were 1:1 and 1:0.1.When no test compounds were added, the amount of vehicle added to thereaction mixture was equal to the amount used to deliver the testcompounds. After 5 days of incubation at 37° C. in the presence ofcompounds or vehicle, CD spectra were recorded on a Jasco 810spectropolarimeter (Easton, Md.). All CD spectra were collected in 0.05or 0.1 cm quartz cells. Wavelength traces were scanned from 190-270 nmat 0.1 nm increments with a bandwidth of 2 nm, at a scan speed of 50 nmper minute, a response time of 1 second, and a data pitch of 0.1 nm. Thewhole system was equilibrated and continuously flushed with nitrogen at10 L/min. For data processing, 10 replicate spectra of Aβ₁₋₄₂ withvehicle added were acquired before incubation, averaged, and subtractedfrom 10 averaged spectra of “Aβ₁₋₄₂+test compound” or vehicle after theincubation period. Average spectra were converted from ellipticity indegrees to specific ellipticity using the formula [Ψ]=(Ψ°/d)xc where Ψ°is the ellipticity in degrees, d is the pathlength in mm and c is theconcentration in mg/ml. In this manner, the change in the structure ofthe peptide that occurs between that found at the time of initialdissolution and that found after incubation can be assessed.

FIG. 1A shows some of the CD spectra generated in this study. Aβ₁₋₄₂alone (vehicle in FIG. 1A) in 20 mM acetate buffer after incubationusually demonstrated the typical CD spectrum of an amyloid protein withsignificant β-sheet structure, as demonstrated by the minimum observedat 218 nm. However, in the presence of some of the compounds, a markeddisruption of the β-sheet structure in Aβ₁₋₄₂ fibrils was evident (witha significant increase in random coil or α-helix) as shown by thereduction in the magnitude of the minimum observed at 218 nm (compare toAβ₁₋₄₂ alone).

FIG. 1B shows the effects of compounds 1 and 2 on inhibition of theβ-sheet structure of Aβ₁₋₄₂ fibril formation when compared to a positivecontrol compound. The CD studies demonstrate that the compounds of thisinvention have the ability to disrupt/disassemble the β-sheet structurecharacteristic of Alzheimer's Aβ fibrils. The results of the studiesalso confirm the previous examples using Thioflavin T fluorometry andCongo red binding type assays.

Example 7 Compounds of this Invention are Potent Disrupters ofParkinson's Disease α-Synuclein Fibrils

The tested compounds of this invention were found also to be potentdisruptors/inhibitors of Parkinson's disease α-synuclein fibrils.α-synuclein has been demonstrated to form fibrils when incubated at 37°C. for several days. α-synuclein is postulated to play an important rolein the pathogenesis of Parkinson's disease and other synucleinopathies.In this set of studies, the efficacy of the compounds to cause adisassembly/disruption of pre-formed α-synuclein fibrils of Parkinson'sdisease was analyzed.

Part A—Thioflavin T Fluorometry

In one study, Thioflavin T fluorometry was used to determine the effectsof the compounds and EDTA (as a negative control, (−C)). In this assay,Thioflavin T binds specifically to α-synuclein fibrils, and this bindingproduces a fluorescence enhancement at 485 nm that is directlyproportional to the amount of α-synuclein fibrils present. The higherthe fluorescence, the greater the amount of α-synuclein fibrils present(Naki et al, Lab. Invest. 65:104-110, 1991; Levine III, Protein Sci.2:404-410, 1993; Amyloid: Int. J. Exp. Clin. Invest. 2:1-6, 1995).

In this study, 30 μL of a 1 mg/mL solution of α-synuclein (rPeptide) waspre-fibrillized at 37° C. with agitation at 1400 rpm for 4 days andsubsequently incubated at 37° C. for 3 days either alone or in thepresence of the compounds or EDTA (at α-synuclein:compound weight ratiosof 1:1, 1:0.1, 1:0.01 or 1:0.001). Following 3-days of co-incubation, 50μl of each incubation mixture was transferred into a 96-well microtiterplate containing 150 μl of distilled water and 50 μl of a Thioflavin Tsolution (i.e. 500 mM Thioflavin T in 250 mM phosphate buffer, pH 6.8).The emission fluorescence was read at 485 nm (444 nm excitationwavelength) using an ELISA plate fluorometer after subtraction withbuffer alone or compound alone, as blank.

The results of the 3-day incubations are graphically illustrated in FIG.7. For example, whereas EDTA caused no significant inhibition ofα-synuclein fibrils at all concentrations tested, all of the compoundscaused a dose-dependent disruption/disassembly of pre-formed α-synucleinfibrils to various extents. For example, at an α-synuclein:compoundratio of 1:0.01 the positive control compound (+C in FIG. 7) caused asignificant (p<0.01) 77.4% inhibition whereas the other compounds testeddisplayed a range from 45 to 83%. Compounds 1, 4, 5 and 6 displayedresults very similar to the positive control compound. This studyindicated that compounds of this invention are potentdisruptors/inhibitors of Parkinson's disease α-synuclein fibrils, andusually exert their effects in a dose-dependent manner.

Part B: Congo Red

In the Congo red binding assay, the ability of a given test compound toalter α-synuclein binding to Congo red is quantified. In this assay,α-synuclein (pre-fibrillized as prepared in the Thio T assay) andcompounds were incubated for 3 days and then vacuum filtered through a0.2 μm filter. The amount of α-synuclein retained in the filter was thenquantitated following staining of the filter with Congo red. Afterappropriate washing of the filter, any lowering of the Congo red coloron the filter in the presence of the compound (compared to the Congo redstaining of the amyloid protein in the absence of the compound) wasindicative of the test compound's ability to diminish/alter the amountof aggregated and congophilic α-synuclein.

In one study, the ability of α-synuclein fibrils to bind Congo red inthe absence or presence of increasing amounts of compounds or EDTA (atα-synuclein:compound weight ratios of 1:1, 1:0.1, 1:0.01 or 1:0.001) wasdetermined. The results of 3-day incubations are graphically illustratedin FIG. 8. Whereas EDTA (−C) caused no significant inhibition ofα-synuclein fibril binding to Congo red at all concentrations tested,the compounds tested caused a dose-dependent inhibition of α-synucleinbinding to Congo red. For example, the positive control compound (+C)caused a significant (p<0.01) 78.5% inhibition of Congo red binding toα-synuclein fibrils at a wt/wt ratio of 1:1. The range of inhibition atthe same ratio for the all of the compounds tested was from 60 to 100%.This study indicated that compounds of this invention are also potentinhibitors of Parkinson's disease type α-synuclein fibril binding toCongo red, and usually exert their effects in a dose-dependent manner.

Part C—Circular Dichroism

Circular dichroism (CD) spectroscopy is a method that can be used todetermine effects of test compounds on disruption of the secondarystructural conformation of α-synuclein fibrils. In one study, asdescribed in this example, circular dichroism spectroscopy was used todetermine the effects of different compounds of the invention on theβ-sheet conformation of α-synuclein fibrils. For this study, α-synuclein(rPeptide Inc., Bogart, Ga.) was dissolved in 9.5 mM phosphate buffer(PBS) to 1 mg/ml. The resulting stock was diluted in the same buffer andeither test compounds or vehicle added such that the final concentrationof peptide was 0.25 mg/ml and the α-synuclein:compound wt/wt ratios were1:1 and 1:0.1. A CD spectrum was recorded of the vehicle treated sampleprior to incubation of all samples for 4 days, after which spectra forall α-synuclein/compound or vehicle reactions were acquired. CD spectrawere recorded on a Jasco 810 spectropolarimeter (Easton, Md.). All CDspectra were acquired using 0.10 cm quartz cells. Wavelength traces werescanned from 190-270 nm at 0.1 nm increments with a bandwidth of 2 nm,at a scan speed of 50 nm/minute, a response time of 32 seconds, and adata pitch of 0.5 nm. The whole system was equilibrated and continuouslyflushed with nitrogen at 10 L/min. For data processing, 10 replicatespectra of the buffer with vehicle added were acquired beforeincubation, averaged, and subtracted from 10 averaged spectra of“α-synuclein+test compound” or vehicle after the incubation period.Average spectra were converted from ellipticity in degrees to specificellipticity using the formula [Ψ]=(Ψ°/d)xc where Ψ° is the ellipticityin degrees, d is the pathlength in mm and c is the concentration inmg/ml. In this manner, the change in the structure of the peptide thatoccurs between that found at the time of initial dissolution and thatfound after incubation can be assessed.

FIG. 2 shows the CD spectra generated for α-synuclein at time zero andafter 4 days of incubation at 37° C. α-synuclein alone in vehicletreated PBS buffer demonstrated the random coil signature at time zeroand after 4 days of incubation demonstrated the typical CD spectrum of aprotein with significant β-sheet structure, as demonstrated by theminimum observed at 218 nm. However, in the presence of some of thecompounds, a marked disruption of the β-sheet structure in α-synucleinfibrils was evident (with a significant increase in random coil orα-helix) as shown by the reduction in the magnitude of the minimumobserved at 218 nm (compare to α-synuclein alone).

FIG. 3A shows some of the CD spectra generated in this study.α-synuclein at time zero produces the spectrum indicative of a randomcoil peptide and also provides the 100% inhibition control data. Afterincubation, the spectrum of α-synuclein is what would be expected for aβ-sheet structure, indicating higher order aggregates have formed and isused to provide the 0% inhibition control data. Samples used for thecontrols are vehicle treated to assure a quantitative relationshipbetween these samples and the test compound treated samples. These twospectra allow for the precise quantitation of the percent inhibition offibril formation in the test compound treated samples due to theirestablishing of the positive and negative controls, which are assumed tobe 100% and 0% fibrillar respectively. Despite these control percentagesbeing only estimates, there is insufficient uncertainty to make themsuspect, ie the lower end may be 0-5% inhibition while the upper end maybe 95-100% inhibition. The controls are generated in each batch run,using the same stock solution of α-synuclein which is fractioned intoaliquots of equal volume to which the individual test compound orvehicle are added and run in parallel to assure the accuracy of thequantitation. The spectra shown in FIG. 3A were acquired with anα-synuclein/test compound ratio of 1:1 wt/wt. These CD spectrademonstrate that the compounds of this invention have the ability todisrupt/disassemble the β-sheet structure characteristic of Parkinson'sdisease α-synuclein fibrils.

FIG. 3B shows the effects of compounds on inhibition of the β-sheetstructure of α-synuclein when compared to a positive control compound(+C). The positive control (+C1) is, as stated, the time zero vehicletreated spectrum while the negative control (−C) is the 4 day vehicletreated spectrum.

FIG. 4A shows some of the CD spectra that were acquired in this study.These spectra were acquired and processed in the same manner as thosepresented in FIG. 3A. These spectra also show marked disruption of theβ-sheet signature found in the spectrum of the vehicle treated sample.

FIG. 4B shows the effects of compounds on inhibition of the β-sheetstructure of α-synuclein when compared to a positive control compound(+C). The positive control (+C1) is, as stated, the time zero vehicletreated spectrum while the negative control (−C) is the 4 day vehicletreated spectrum.

The results of the studies also confirm the previous examples usingThioflavin T fluorometry and Congo red binding type assays, that thecompounds of this invention are potent anti-α-synuclein fibrilizationagents.

Example 8 Compounds of this Invention are Potent Disruptors/Inhibitorsof α-Synuclein Fibrils Associated with Parkinson's Disease

Parkinson's Disease is characterized by the accumulation of insolubleintraneuronal aggregates called Lewy Bodies, a major component of whichis α-synuclein (reviewed in Dauer et al., Neuron, 39:889-909, 2003).Since autosomal dominant mutations in α-synuclein cause a subset ofcases of familial Parkinson's disease, and since these mutationsincrease the likelihood of α-synuclein to aggregate and form LewyBodies, aggregated α-synuclein is proposed to be directly involved inthe etiology and disease progression (Polymeropoulos et al., Science276:1197-1199, 1997; Papadimitriou et al., Neurology 52:651-654, 1999).Structural studies have revealed that intracellular Lewy bodies containa large proportion of misfolded proteins with a high degree of β-pleatedsheet secondary structure. These studies were conducted to determine theefficacy of the test compounds in the inhibition/disruption ofα-synuclein fibrils associated with Parkinson's disease.

Therefore, to test the therapeutic potential of the compounds, twocell-based assays were utilized. In both assays, rotenone is used toinduce mitochondrial oxidative stress and α-synuclein aggregation. Thefirst assay utilizes the binding of the fluorescent dye thioflavin S tostructures with high β-sheet content including α-synuclein fibrils.Therefore, quantitative assessment of the extent of thioflavinS-positive staining of fixed cells is used to test the ability of thecompounds to decrease the amount of α-synuclein aggregates. In thesecond assay, cell viability is assessed using the XTT Cytotoxicityassay, which is dependent on intact, functional mitochondria in livecells. Thus, the XTT Cytotoxicity assay is used to test the ability ofthe compounds to ameliorate the mitochondrial toxicity and resultingloss of viability associated with the accumulation of α-synucleinaggregates. Phrased another way, the XTT Cytotoxicity assay is used togauge the compounds neuroprotective efficacy. These studies arepresented in the following examples.

To carry out these studies, a cell culture model was used in which humanα-synuclein aggregation is experimentally induced. BE-M17 humanneuroblastoma cells stably transfected with A53T-mutant humanα-synuclein were obtained. Cell culture reagents were obtained fromGibco/Invitrogen, and cells were grown in OPTIMEM supplemented with 10%FBS, Penicillin (100 units/ml), Streptomycin (100 μg/ml) and 500 μg/mlG418 as previously described (Ostrerova-Golts et al., J. Neurosci.,20:6048-6054, 2000).

Thioflavin S is commonly used to detect amyloid-containing structures insitu, including in brain tissue (Vallet et al., Acta Neuropathol.,83:170-178, 1992), and cultured cells (Ostrerova-Golts et al., J.Neurosci., 20:6048-6054, 2000), whereas thioflavin T is often used as anin vitro reagent to analyze the aggregation of soluble amyloid proteinsinto fibrils enriched in β-pleated sheet structures (LeVine III, Prot.Sci., 2:404-410, 1993). Therefore, Thioflavin S histochemistry was usedon cultured cells to detect aggregates containing a high degree ofβ-pleated structures that formed in response to oxidativestress-inducing agents (in this case rotenone) as previously described,with minor modifications (Ostrerova-Golts et al., J. Neurosci.,20:6048-6054, 2000). Briefly, for these studies, cells were grown onPoly-D-Lysine coated glass slide chambers at approximately 3×10⁴cells/cm². After 24 hours, cells were treated with 500 nM, 1 μM or 5 μMrotenone (Sigma) or vehicle (0.05% DMSO) as indicated. Immediately afterrotenone (or vehicle) addition, compounds were added at the indicatedconcentration, or cell culture media only (no compound) in the presenceof rotenone was added. Identical treatments were repeated after 48hours. After an additional 48 hours, cells were fixed for 25 minutes in3% paraformaldehyde. After a PBS wash, the cells were incubated with0.015% thioflavin S in 50% ethanol for 25 minutes, washed twice for fourminutes in 50% ethanol and twice for five minutes in deionized water andthen mounted using an aqueous-based mountant designed to protect againstphotobleaching. Aggregates that bind to thioflavin S were detected witha fluorescent microscope using a High Q FITC filter set (480 to 535 nmbandwidth) and a 20× objective lens unless otherwise indicated. Between8 and 16 representative images per condition were selected, imaged andprocessed by an experimenter who was blinded to treatment conditions. Toassess the amount of thioflavin S-positive aggregates, the total areaper field covered by thioflavin S-positive inclusions was determined.For this purpose, background fluorescence that failed to exceed pre-setsize or pixel intensity threshold parameters was eliminated usingQ-capture software. Spurious, non-cell associated fluorescence wasmanually removed. Unless indicated otherwise, data represent groupmeans±SEM. Statistical analyses were performed with GraphPad Prism(GraphPad Inc). Differences between means (two samples) were assessed bythe Student's t test. Differences among multiple means were assessed byone-factor ANOVA followed by Tukey's multiple comparison test.

To validate the ability of the assay to quantitatively detect aggregatesthat bind thioflavin S, staining of BE-M17 cells overexpressing A53Tα-synuclein was carried out and the results revealed a rotenonedose-dependent increase in thioflavin S-positive aggregates relative tovehicle-treated control cells (FIG. 9A-D). Higher magnification imagesobtained with a 40× objective indicated that the thioflavin S-positiveaggregates were intracellular and cytoplasmic (FIG. 9D), analogous tothe accumulation of intracytoplasmic Lewy bodies which are pathologicalhallmarks associated with Parkinson's disease. Quantitation of the areacovered by thioflavin-S-positive aggregates established that 5 μM ofrotenone was sufficient to induce robust aggregation (FIG. 9E) and thusis an effective dose to test the ability of compounds to attenuate theformation of these aggregates.

Using the protocol described above, several compounds were tested fortheir ability to reduce, prevent or eliminate thioflavin S-positiveaggregates in rotenone-treated BE-M17 cells overexpressing A53Tα-synuclein. Examples of results obtained from experiments using thesecompounds are described below.

In cells treated with 1 μM rotenone only, there was a robust presence ofthioflavin S-positive aggregates (FIG. 10A). Addition of 500 ng/ml (FIG.10B) or 1 μg/ml (FIG. 10C) of positive control compound markedly reducedthe abundance of these rotenone-induced aggregates by 87% and 91%respectively (as shown in FIG. 10D) relative to rotenone only-treatedcells. Therefore, the positive control compound is highly effective atthe reduction, prevention and/or elimination of thioflavin S-positiveaggregates in cells that express human A53T α-synuclein.

Addition of from 500 ng/ml up to 2 μg/ml (FIGS. 11B-D) of compound 1 didnot reduce the abundance of rotenone-induced aggregates relative torotenone only-treated cells (FIGS. 11D and E).

As shown in FIG. 12, in cells treated with 1 μM rotenone the addition of500 ng/ml and 2 μg/ml of compound 2 reduced the abundance ofrotenone-induced aggregates by 39-44%, and in cells treated with 5 μMrotenone the addition of 1 μg/ml of compound 2 markedly reduced theabundance of rotenone-induced aggregates by 67% (FIG. 12E).

FIGS. 13 A-E show the effects of compound 3. In cells treated with 1 μMrotenone the addition of 500 ng/ml up to 2 μg/ml of compound 3 reducedthe abundance of rotenone-induced aggregates by 41 to 63% relative torotenone only-treated cells.

Addition of 500 ng/ml up to 2 μg/ml of compound 4 did not reduce theabundance of rotenone-induced aggregates relative to rotenoneonly-treated cells (FIGS. 14A-E).

FIGS. 15 A-E show the effects of compound 5. In cells treated with 1 μMrotenone the addition of 1-2 μg/ml of compound 3 reduced the abundanceof rotenone-induced aggregates by 25 to 49% relative to rotenoneonly-treated cells.

The addition of compound 6 did not have significant effects on theabundance of rotenone-induced aggregates relative to rotenoneonly-treated cells (FIGS. 16A-E).

The addition of 500 ng/ml and 2 μg/ml of compound 7 markedly reduced theabundance of rotenone-induced aggregates by 60 and 74% (respectively)relative to rotenone only-treated cells in cells treated with 1 μMrotenone only. In cells treated with 5 μM rotenone the addition of 500ng/ml and 2 μg/ml of compound 7 reduced the abundance ofrotenone-induced aggregates by 31 and 67% (respectively) (FIGS. 17A-E).

FIGS. 18 A-E show the effects of compound 8. In cells treated with 1 μMrotenone the addition of 1 μg/ml of compound 8 reduced the abundance ofrotenone-induced aggregates by 56% relative to rotenone only-treatedcells. In cells treated with 5 μM rotenone the addition of 1 or 2 μg/mlof compound 8 reduced the abundance of rotenone-induced aggregates by 48and 38% (respectively).

The addition of 500 ng/ml up to 2 μg/ml of compound 9 reduced theabundance of rotenone-induced aggregates from 19 to 60% relative torotenone only-treated cells in cells treated with 1 μM rotenone (FIGS.19A-E). The addition of compound 9 did not reduce the abundance ofrotenone-induced aggregates in cells treated with 5 μM rotenone althoughbaseline staining was lower than expected at this rotenone dose.

In conclusion, many of the compounds tested, especially compounds 2, 3,5, 7, 8 and 9 effectively and potently reduced, prevented, inhibitedand/or eliminated the formation, deposition and/or accumulation ofα-synuclein aggregates in A53T α-synuclein-expressing BE-M17 cells.

Example 9 Compounds of this Invention Protect Against Rotenone-InducedCytotoxicity

The XTT Cytotoxicity Assay (Roche Diagnostics, Mannheim, Germany) waspreviously used to demonstrate that A53T α-synuclein potentiates celldeath in BE-M17 cells through an oxidative stress-dependent mechanism(Ostrerova-Golts et al., J. Neurosci., 20:6048-6054, 2000). Research hasshown that the accumulation of α-synuclein fibrils in Lewy bodiescontributes mechanistically to the degradation of neurons in Parkinson'sdisease and related disorders (Polymeropoulos et al., Science276:2045-2047, 1997; Kruger et al., Nature Genet. 18:106-108, 1998).Here, the XTT Cytotoxicity assay (hereafter referred to as the XTTassay) was used to measure the ability of test compounds to protectagainst rotenone-induced cytotoxicity (neuroprotective ability). Theassay is based on the principle that conversion of the yellowtetrazolium salt XTT to form an orange formazan dye (that absorbs lightaround 490 nm) occurs only in metabolically active, viable cells.Therefore, light absorbance at 490 nm is proportional to cell viability.For this assay, cells were plated in 96 well tissue culture dishes at10⁴ cells per well. After 24 hours, cells were treated with 500 nM or 2μM rotenone, or vehicle (0.05% DMSO) as indicated. Immediately afterrotenone addition, compounds were added at the indicated concentration.As a control, compounds were added without rotenone (vehicle only, 0.05%DMSO) and resulted in no toxicity at the doses tested. Untreated cellsreceived cell culture media only (no compound, with or withoutrotenone). After 40-44 hours of treatment, conditioned media was removedand replaced with 100 μl fresh media and 50 μl XTT labeling reactionmixture according to the manufacturer's recommendations. Five to sixhours later, the absorbance at 490 nm was measured and corrected forabsorbance at the 700 nm reference wavelength. Treatment with 500 nM and2 μM rotenone usually decreased viability by 35-45% relative tountreated cell without rotenone (FIG. 20). Percent inhibition of celldeath was calculated as the proportion of the rotenone-inducedabsorbance (viability) decrease that was eliminated by test compoundtreatment.

Treatment with positive control compound at 10-25 μg/ml inhibited therotenone-induced loss of viability by 25-33% at both rotenone doses(FIG. 21).

The experiment was performed with each compound and the results areshown in FIGS. 22-30. FIGS. 22-30, panel A graphically illustrates thetoxicity of the compound whereas FIGS. 22-30, panel B show inhibition bythe compound of the rotenone-induced loss of viability measured at bothrotenone doses.

Treatment with 10 μg/ml of compound 1 indicates that this compound isnon-toxic, whereas higher doses displayed some toxicity (FIG. 22A).Treatment with 10 μg/ml of compound 1 inhibited the rotenone-inducedloss of viability by approximately 18 to 27% at both rotenone doses(FIG. 22B).

Treatment with 10-25 μg/ml of compound 2 indicates that this compound isnon-toxic, whereas a dose of 50 μg/ml displayed some toxicity (FIG.23A). Treatment with 25 μg/ml of compound 2 inhibited therotenone-induced loss of viability by approximately 20 to 28% at bothrotenone doses (FIG. 23B).

Treatment with 10 to 50 μg/ml of compound 3 indicates that this compoundis relatively non-toxic at all the doses tested (FIG. 24A). Treatmentwith 10 to 50 μg/ml of compound 3 inhibited the rotenone-induced loss ofviability by approximately 17 to 28% at both rotenone doses (FIG. 24B).

Treatment with 10 and 25 μg/ml of compound 4 indicates that thiscompound is non-toxic, whereas a dose of 50 μg/ml displayed minimaltoxicity (FIG. 25A). Treatment with 25 μg/ml of compound 4 wasparticularly effective and inhibited the rotenone-induced loss ofviability by approximately 50% at the 500 nM dose of rotenone, whereasat 2 μM the inhibition observed was approximately 26% (FIG. 25B).

Treatment with 10 or 25 μg/ml of compound 5 indicates that this compoundis non-toxic, whereas a dose of 50 μg/ml displayed minimal toxicity(FIG. 26A). Treatment with compound 5 did not cause any inhibition ofthe rotenone-induced loss of viability at either rotenone dose (FIG.26B).

Treatment with 10 to 50 μg/ml of compound 6 indicates that this compoundis non-toxic at all the doses tested (FIG. 27A). Treatment with 50 μg/mlof compound 6 at the 500 nM dose of rotenone inhibited therotenone-induced loss of viability by approximately 10% whereas at 2 μMrotenone, the inhibition observed was approximately 12-16% for all dosesof the compound tested (FIG. 27B).

Treatment with 1 to 50 μg/ml of compound 7 indicates that this compoundis non-toxic, and even higher doses (100-150 μg/ml) displayed only veryminimal toxicity (FIG. 28A). Treatment with 50 μg/ml of compound 7showed the highest inhibition of the rotenone-induced loss of viabilityat approximately 25% at the 500 nM dose of rotenone (FIG. 28B).

Treatment with 10 to 25 μg/ml of compound 8 indicates that this compoundis relatively non-toxic (FIG. 29A). Despite some minor toxicity at 50μg/ml, the compound apparently affords some protection against thestrong rotenone toxicity. This conclusion is supported by morphologicalanalysis (not shown). Treatment with 50 μg/ml of compound 1 at 2 μMrotenone showed inhibition of the rotenone-induced loss of viability byapproximately 18% (FIG. 29B).

Treatment with 10 to 25 μg/ml of compound 9 indicates that this compoundis relatively non-toxic, whereas the higher dose displayed some toxicity(FIG. 30A). Treatment with compound 9 did not cause any appreciableinhibition of the rotenone-induced loss of viability at either rotenonedose (FIG. 30B).

In conclusion, many of the tested compounds were efficacious ininhibiting rotenone-induced cytotoxicity demonstrating neuroprotectiveactivity against α-synuclein toxicity.

Example 10 Improved Motor Performance of α-Synuclein Transgenic MiceTreated with Compounds of this Invention

To assess the potential efficacy of compounds in a Parkinson'sdisease-relevant mouse model, transgenic mice overexpressing wild-typehuman α-synuclein under the control of the mouse Thy-1 promoter(Rockenstein E, et al., 2002. J Neurosci Res 68:568-578) were used.Human α-synuclein transgenic mice have proven to be useful models forParkinson's disease, and thus a suitable system for testing potentialtherapeutic agents, for a number of reasons including the following. (1)The presence of α-synuclein aggregates that are detectable by bothimmunohistochemical (staining) and biochemical (western blot) methods.These aggregates are similar to the Lewy bodies (intracellularinclusions comprised primarily of α-synuclein) that are the pathologicalhallmark of Parkinson's disease (Rockenstein E, et al., 2002. J.Neurosci. Res. 68:568-578 and Hashimoto M, et al., 2003 Ann N Y Acad Sci991:171-188). (2) The mice experience a dopaminergic deficit in thenigrostriatal pathway, as indicated by loss of tyrosinehydroxylase-immunoreactive neuronal projections in the striatum(Hashimoto M, et al., 2003 Ann N Y Acad Sci 991:171-188). This deficitis also seen in human PD patients. (3) The mice show deficits, includingslowness of movement, loss of balance and coordination and muscleweakness in a motor function-dependent behavioral test such as the beamtraversal test (Fleming S M, et al., 2004 J Neurosci 24:9434-9440 andFleming S M, et al., 2006 Neuroscience 142:1245-1253).

Similar motor dysfunction is seen in human PD patients. To assess thepotential efficacy of compounds to improve motor performance or minimizedeficites, the challenging beam traversal test was conducted oncompound-treated and vehicle-treated mice assessed prior to treatment at0 months and again at 3 and 6 months of treatment. If compounds wereeffective, one would expect that mice administered these compounds wouldperform better than vehicle-treated mice at the same age, and/or thattest compound treatment might ameliorate age-dependent decline inperformance within a given group. For example, if test compounds wereeffective, one might expect compound treated-mice to cross the beam morequickly, relative to vehicle-treated mice. Or one might expectage-dependent impairments within a group to be lessened (for exampleperformance after compound treatment might be similar to performanceprior to treatment, or even better, whereas vehicle-treated mice performprogressively worse over the same period of time).

Beam Traversal Test

In the beam traversal test, which is one measure of motor performance,mice are trained over two days, with five trials per day, to cross anarrowing beam (separated into four segments) with support ledgesattached along each side, and leading to the animal's home cage. On thethird day, the test is made more challenging by placing a mesh grid overthe beam surface, leaving a small space of about 1 cm between the gridand the surface of the beam. Animals are then videotaped over a periodof five trials, and the time to cross, number of steps taken and numberof slips are recorded by an investigator blind to drug treatment(Fleming S M, et al., 2006. Neuroscience 142:1245-1253).

Transgenic mice administered compound 2 for three months showed amarked, significant 49% improvement (time to cross the beam) relative tovehicle-treated, age-matched (15 months of age), control mice (FIG. 31).After six months of treatment, however, performance was similar tovehicle-treated mice at this age (FIG. 31). Taken together, these datashow that compound 2 delays the onset of behavioral deficits in the beamtraversal test.

Transgenic mice administered compound 7 for six months showed a marked,significant 35% improvement (time to cross the beam) relative tovehicle-treated, age-matched (15 months of age), control mice (FIG. 31).In addition, after only three months of compound 7 treatment,performance was 39% improved relative to vehicle-treated controls (FIG.31). Taken together, these data show that compound 7 treatment preventsthe age-dependent progression of deficits in the beam traversal test.

Example 11 Compositions of Compounds of this Invention

The compounds of this invention, as mentioned previously, are desirablyadministered in the form of pharmaceutical compositions. Suitablepharmaceutical compositions, and the method of preparing them, arewell-known to persons of ordinary skill in the art and are described insuch treatises as Remington: The Science and Practice of Pharmacy, A.Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins,Philadelphia, Pa.

Representative compositions are as follows:

Oral Tablet Formulation

An oral tablet formulation of a compound of this invention is preparedas follows:

% w/w Compound of this invention 10.0 Magnesium stearate 0.5 Starch 2.0Hydroxypropylmethylcellulose 1.0 Microcrystalline cellulose 86.5

The ingredients are mixed to homogeneity, then granulated with the aidof water, and the granulates are dried. The dried granulate is thencompressed into tablets sized to give a suitable dose of the compound.The tablet is optionally coated by applying a suspension of a filmforming agent (e.g. hydroxypropylmethylcellulose), pigment (e.g.titanium dioxide), and plasticizer (e.g. diethyl phthalate), and dryingthe film by evaporation of the solvent. The film coat may comprise, forexample, 2-6% of the tablet weight.

Oral Capsule Formulation

The granulate from the previous section of this Example is filled intohard gelatin capsules of a size suitable to the intended dose. Thecapsule is banded for sealing, if desired.

Softgel Formulation

A softgel formulation is prepared as follows:

% w/w Compound of this invention 20.0 Polyethylene glycol 400 80.0

The compound is dissolved or dispersed in the polyethylene glycol, and athickening agent added if required. A quantity of the formulationsufficient to provide the desired dose of the compound is then filledinto softgels.

Parenteral Formulation

A parenteral formulation is prepared as follows:

% w/w Compound of this invention 1.0 Normal saline 99.0

The compound is dissolved in the saline, and the resulting solution issterilized and filled into vials, ampoules, and prefilled syringes, asappropriate.

Controlled-Release Oral Formulation

A sustained release formulation may be prepared by the method of U.S.Pat. No. 4,710,384, as follows:

One Kg of a compound of this invention is coated in a modified Uni-Glattpowder coater with Dow Type 10 ethyl cellulose. The spraying solution isan 8% solution of the ethyl cellulose in 90% acetone to 10% ethanol.Castor oil is added as plasticizer in an amount equal to 20% of theethyl cellulose present. The spraying conditions are as follows: 1)speed, 1 liter/hour; 2) flap, 10-15%; 3) inlet temperature, 50° C., 4)outlet temperature, 30° C., 5) percent of coating, 17%. The coatedcompound is sieved to particle sizes between 74 and 210 microns.Attention is paid to ensure a good mix of particles of different sizeswithin that range. Four hundred mg of the coated particles are mixedwith 100 mg of starch and the mixture is compressed in a hand press to1.5 tons to produce a 500 mg controlled release tablet.

The present invention is not limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing descriptions. Such modificationsare intended to fall within the scope of the appended claims. Variouspublications are cited herein, the disclosures of which are incorporatedby reference in their entireties.

1. A compound selected from the group consisting of:

where R₁, R₂, R₃, and R₄ are independently positioned hydroxyl groupsand R is selected from —C(O)NR′, sulfonamide, heteroaryl, tricycloalkylor pharmaceutically acceptable esters or salts thereof and where R′ isselected from H or CH₃, and such that when R is —C(O)NR′ and one ofeither the R₁ and R₂ hydroxyl groups or the R₃ and R₄ hydroxyl groupsare at the 3,4 position, then the other hydroxyl groups are at one ofthe positions selected from the group consisting of 2,3; 2,4; 2,5; 2,6;3,5; 3,6; 4,5; 4,6 and 5,6.
 2. The compound of claim 1 where R is—C(O)NR′ and R′ is selected from H or CH₃ or pharmaceutically acceptablesalts thereof.
 3. The compound of claim 1 where R′ is H.
 4. The compoundof claim 1 selected from the group consisting of: 2,3-dihydroxybenzoicacid 3,4-dihydroxyanilide; 3,4-dihydroxybenzoic acid2,3-dihydroxyanilide; and 2,3-dihydroxybenzoic acid2,3-dihydroxyanilide.
 5. The compound of claim 1 where R′ is CH₃.
 6. Thecompound of claim 1 where the compound is 3,4 dihydroxybenzoic acid 3,4dihydroxy N-methyl anilide.
 7. The compound of claim 1 where R issulfonamide.
 8. The compound of claim 1 where the compound is3,4-dihydroxybenzenesulfonic acid 3,4-dihydroxyphenylsulfonamide.
 9. Thecompound of claim 1 where R is heteroaryl.
 10. The compound of claim 1where the compound is selected from the group consisting of imidazole,triazole, and pyrazole.
 11. The compound of claim 1 where the compoundis selected from the group consisting of:2,4-bis(3,4-dihydroxyphenyl)imidazole; 3,5-bis(3,4 dihydroxyphenyl)1,2,4 triazole; and 3,5-bis(3,4 dihydroxyphenyl)pyrazole.
 12. Thecompound of claim 1 where R is tricycloalkyl.
 13. The compound of claim1 where the compound is 1,3 bis-(3,4 dihydroxyphenyl)adamantane.
 14. Apharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable excipient.
 15. A method of treating theformation, deposition, accumulation, or persistence of Aβ amyloid orα-synuclein fibrils, comprising treating the fibrils with an effectiveamount of the compound of claim
 1. 16. The method of claim 15 where thecompound is selected from the group consisting of (1) the compounds thatare: 2,3-dihydroxybenzoic acid 3,4-dihydroxyanilide,3,4-dihydroxybenzoic acid 2,3-dihydroxyanilide, 2,3-dihydroxybenzoicacid 2,3-dihydroxyanilide, 3,4-dihydroxybenzoic acid 3,4-dihydroxyN-methyl anilide, 3,4-dihydroxybenzenesulfonic acid3,4-dihydroxyphenylsulfonamide, 2,4-bis(3,4 dihydroxyphenyl)imidazole,3,5-bis(3,4 dihydroxyphenyl) 1,2,4 triazole, 3,5-bis(3,4dihydroxyphenyl)pyrazole, and 1,3-bis(3,4 dihydroxyphenyl)adamantane;(2) the methylenedioxy analogs and pharmaceutically acceptable estersthereof, and (3) the pharmaceutically acceptable salts of the compoundsof (1) and (2).
 17. A method of treating a β-amyloid disease or asynucleinopathy in a mammal suffering therefrom, comprisingadministration of a therapeutically effective amount of the compound ofclaim
 1. 18. The method of claim 17 where the β-amyloid disease isselected from the group of diseases consisting of Alzheimer's disease,Down's syndrome, hereditary cerebral hemorrhage with amyloidosis of theDutch type, and cerebral β-amyloid angiopathy.
 19. The method of claim17 where the β-amyloid disease is Alzheimer's disease.
 20. The method ofclaim 17 where the synucleinopathy is selected from the group consistingof Parkinson's disease, familial Parkinson's disease, Lewy body disease,the Lewy body variant of Alzheimer's disease, dementia with Lewy bodies,multiple system atrophy, and the Parkinsonism-dementia complex of Guam.21. The method of claim 17 where the synucleinopathy is Parkinson'sdisease.
 22. The method of claim 17 where the compound is selected fromthe group consisting of (1) the compounds that are: 2,3-dihydroxybenzoicacid 3,4-dihydroxyanilide, 3,4-dihydroxybenzoic acid2,3-dihydroxyanilide, 2,3-dihydroxybenzoic acid 2,3-dihydroxyanilide,3,4-dihydroxybenzoic acid 3,4-dihydroxy N-methylanilide,3,4-dihydroxybenzenesulfonic acid 3,4-dihydroxyphenylsulfonamide,2,4-bis(3,4 dihydroxyphenyl)imidazole, 3,5-bis(3,4 dihydroxyphenyl)1,2,4 triazole, 3,5-bis(3,4 dihydroxyphenyl)pyrazole, and 1,3-bis(3,4dihydroxyphenyl)adamantane; (2) the methylenedioxy analogs andpharmaceutically acceptable esters thereof, and (3) the pharmaceuticallyacceptable salts of the compounds of (1) and (2).